The document discusses three research proposals submitted by the author to the ICAR-Indian Veterinary Research Institute over the last five years related to protecting holy cows. The proposals focused on (1) evaluating new therapies for brucellosis in cattle and buffaloes, (2) surveying infectious diseases in shelters for stray cows, and (3) studying epidemiological factors associated with reduced milk production from communicable diseases.

1. The presentation relates to my three rejected
research proposals, aimed at Protection of Holy cow,
at ICAR-ICAR-Indian Veterinary Research Institute,
Izatnagar-243 122, India, in last five years
DOI: 10.13140/RG.2.2.35372.44161
Available at:
https://www.researchgate.net/publication/371348898_My_research_proposal_rejected_by_the_ICAR-
IVRI_in_the_last_5_years
• Clinical evaluation of newly advocated therapies for brucellosis
in cattle and buffaloes. Duration: September 2019 to August 2021
• A cross sectional survey of Holy Cow Infectious Problems in
Gaushalas (Gaushalas are protective shelters for stray cows in
India). Duration: September 2022-August 2024
• Explorative study on Epidemiological determinants associated
with drastic reduction in Milk Production of Dairy Animals with
reference to communicable diseases . Duration: September 2022-
August 2024

2. Clinical evaluation of newly
advocated therapies for brucellosis
in cattle and buffaloes
Duration: 1st Sept 2019 to 31st August 2021
PI: BR Singh
Co-PIs
1. VK Chaturvedi, PS, BP Division
2. Bablu Kumar, Sr. S, BP Division
3. Dr. DK Sinha, PS, Epidemiology
4. Dr. Vinodh Kumar OR, SS, Epidemiology
5. Dr. DK Singh, PS, VPH
6. Dr. Mayank Rawat, PS Standardization
7. I/c LPM or his Nominee
8. JD CADRAD or his Nominee

3. Brucella- as an intracellular parasite
• Brucella spp. are facultative intracellular pathogens infecting and
replicating inside cells of the mononuclear phagocytic system (MPS),
mainly in the liver and the spleen. After being captured by phagocytic cells
they subvert the regular phagosome maturation process. After ingestion, the
pathogen localizes in early phagosomes. Most of them in vacuoles retaining
late endosomal/ lysosomal markers (LAMP-1 positive) get killed.
However, a few phagocytic vacuoles do not fuse with lysosomes. Brucella
LPS and cyclic beta-1,2-glucan play a role in the control of phagosomal
maturation. Cyclic beta-1,2-glucans, structurally related to cyclodextrins,
can selectively extract and incorporate cholesterol,
glycosylphosphatidylinositol and ganglioside GM-1, which are essential for
bacterial survival and replication, from lipid rafts to phagosomes. Those
vacuoles that successfully evade lysosomal fusion are characterized by the
progressive exclusion of LAMP-1 and are capable of interacting with ER
exit sites. Subsequently, they fuse to ER to generate ER-derived replicative
Brucella-containing vacuoles. In this case, the acidification of the Brucella-
containing vacuole is a requisite for intracellular replication of the bacteria
(Arellano-Reynoso et al., 2005; Starr et al., 2008)

4. Brucellosis
• Endemic in India: 5% to 13.5% of cattle and 3% of buffaloes (Rahman, 2013,
Renukaradhya et al., 2002) are positive for brucellosis.
• Estimated direct loss due to Brucellosis in 2015 was the highest among all livestock
diseases to the tune of Rs. 20400 Krores ( Singh et al., 2015).
• With ELISA 34% of Mithuns in Nagaland (Rajkhowa et al., 2004) and 23.3% Yak
(Bandopadhyay et al., 2008) were positive for Brucellosis.
• >8.5% Sheep in Rajasthan & Bihar were positive for Brucellosis (Singh, 2007).
• In Gujarat 33.7% of herds and 11.5% of animals positive for Brucellosis (Patel, 2014).
• In Maharashtra, with ELISA, >40% of animals were positive for Brucellosis (Lodhe et
al., 2011).
• 22.3% of animals in Haryana and 34.2% of animals in Punjab were positive for
Brucellosis (Chand, 2013).
• Seroprevelence study of bovine brucellosis using indirect- ELISA showed 45.80%,
22.39%, and 8.57% seropositivity in Karnataka, Uttar Pradesh & Uttarakhand,
respectively. (Jagapur, 2013).
• Overall seroprevalence of brucellosis in sheep (26.55%) and goats (10.96%) in UP
(Sinha et al., 2018).
• Imporat Zoonosis: >500000 cases. Gujarat, sero-prevalence of brucellosis in humans
varied with occupation, seropositivity with IELISA was 14.28%, 35.0%, 7.31% and
6.0% in veterinary officers, para-veterinarians, other staff related with animal
husbandry activities and patient with PUO, respectively. (Padher et al., 2018)
• Karnataka: seroprevalence of brucellosis in humans 5.1% (Patil et al., 2016).

5. 402 247 199 122 243 91 232 43 109 49 9 27 2
10567
3750 3405
2843 2622 2615
1942
1336 1361 1121 920 764 700
3.8 6.6 5.8 4.3 9.3 3.5 12 3.2 8 4.4 1 3.5 0.3
0
2000
4000
6000
8000
10000
12000
Positive Sampletested Percentage
Renukaradhya et al., 2002
Brucellosis as seen by PDADMAS

6. Brucellosis in Western UP
Upadhyay et al., 2007
6 6
18
89
17
34 23
373
1 2 3
47
0 0 1
63
16.67 33.33
16.67
52.81
0 0 4.35
16.89
0
50
100
150
200
250
300
350
400
Sampletested Positive Percentage

7. Biannual village
level screening
of pooled milk
samples. For
differentiation
in clean and non
clean herds
Biannual
B.abortus S-19
vaccination for
female calves of
4-8 M age
NATIONAL CONTROL PROGRAMME
ON BRUCELLOSIS
Mass screening
& castration of
infected bulls

8. Reduce the impact of disease on human health and to
reduce economic losses.
NCPB is a time bound 5-year intensive location targeted control
program.
Intends to involve village milk cooperatives in diagnosis and
control through vaccination.
Periodical surveillance using milk ring test for pooled milk and
ELISA for random or herd screening.
Targets B. abortus S19 vaccination for all the female calves of 4 to
8 months in infected villages.
Program assures very high & sustained cost benefit ratio to
farmer & dairy industry & helps to establish accredited herds/
villages.
Expected benefits of NCPB

9. Why the NCPB in India have high
probability for failure?
1. None of the available Brucella vaccines is >60-70% effective (2019, MSU).
2. No solid plan for disposal of Brucella positive animals.
3. No administrative control on animal rearing, management and movement
can be implemented.
4. Farmers hardly know about NCPB and its benefits.
5. Failure of vaccine and vaccination programme (even on well organized dairy
herds in National Institutes brucellosis is rampant despite of regular
vaccination).
6. Brucella has a wide host range, just screening and vaccination of dairy
animals can not be sufficient. Sheep, goat, pigs and other animals are often
reared by the farmers in the same village in the same locality.
7. Dearth of assured and accredited (ICAR-IVRI?) quality semen.
8. Poverty, lack of education and inability to opt for hygiene are at the top of
every plan to fall.

10. Despite NCPB no control of
Brucellosis in the near future.
Then what is next?
Treatment????
Brucella is an intra-cellular pathogen
that often gets localized leaving only
very few options for antibiotic
therapy.

11. Antimicrobials for intracellular pathogens
• Antibiotics: Tetracycline/ doxycycline (not effective against most B. abortus
isolates from our farm) and rifampicin for 45 days is only the oral therapy for
treatment of human brucellosis but rifampicin can not be used in animals being
reserved for human use only. The option left is tetracycline/ chloramphenicol with
our without aminoglycosides.
• Nanoparticles: Polymeric nanoparticles prepared using PEO-b-PtBA diblock
copolymer and carrying streptomycin and doxycycline used for brucellosis
treatment as alternative systems to PLGA copolymers, B. melitensis infected mice
were treated with 9 mg/kg streptomycin and 1.8 mg/kg doxycycline, incorporated
in nanoparticles, after 3 days significant reduction the infection in liver and spleen
(Seleem et al. 2009).
• Amphiphilic antimicrobial peptides (AMPs) penetrates mammalian cells as
cathelicidin LL-37, temporin is bactericidal against intracellular MRSA, α-helical
antimicrobial peptide eCATH1 killed Rhodococcus equi in macrophages, plectasin,
an AMP derived from the pezizalean fungus Pseudoplactenia nigrella is effective
against intracellular S. aureus in human and mouse monocytes.
• Antisense oligonucleotide (AS-ODN)-based technology is a strategy designed to
control gene expression at the RNA level. AS-ODNs are short oligomers (10–30
residues) of nucleic acids or nucleic acid mimics typically complementary to the
target mRNA of genes essential for the survival of the bacteria. Hybridization of
AS-ODN to the target mRNA can inhibit translation, resulting in the repression of
gene expression. To improve delivery into bacterial or mammalian cells, AS-ODNs
are often attached to cell-penetrating peptides (CPP).
• Homeopathy: Bacillinum and Arsenicum iodatum
• Ayurvedic/ herbal antimicrobials

12. Antibiotic entry inside cells
• Small (< 700 Da in size) lipophilic antibacterials as β-lactams, macrolides and quinolones
enter mammalian cells via diffusion across the lipid bilayer.
• Uptake via endocytosis of large molecules those not diffuse across the membrane.
• Drugs that enter host cells may subsequently be removed by efflux or exocytosis, and made
unable to reach the pathogens.
• The specific intracellular location (vesicle or cytosol) may bring additional challenges to
antibiotics, acidified phagosomes (pH 4 -5) and antibacterials must resist pH insult to be
effective. Only rifamipin is active at pH <5 and to some extent tetracycline too. Other
antibiotics going in cells act at pH 6-7 include β-Lactams,
Aminoglycosides,Chloramphenicol, Erythromycin and Fluoroquinolones. Aminogycoside do
not enter in required concentration in all cells but can enter in mouse, guinea pig, peritoneal
macrophages and rat embryo fibroblasts.
• Many a times of the bacteria inside cells persist in non-replicating phase thus most of the β-
lactams fail because they act only on replicating bacteria.
– Aminoglycosides enter cells via endocytosis, they bind to megalin, the endocytic
receptor expressed in the renal proximal tubule, accumulation in the kidney can cause
nephrotoxicity.
– Quinolones, rifamycins and sulfamethoxazole-trimethoprim are effective against
intracellular bacteria. Oxazolidinones, macrolides and lincosamides also exert
intracellular antibiotic activity.

13. Antibiotics and their intracellular reach
(Singh, 2019)
Antibiotic Group Antibiotic MIC for S. aureus Cellular/extracellular
(C/E) ratio of the drug
Aminoglycosides Gentamicin 0.125–2 <1 (Lysosomes)
Tetracyclines Tetracycline, doxycycline 0.125–1 0.032–0.5 1.8–7.1
Macrolides Erythromycin
Azithromycin
Clarithromycin
0.064–1
0.25–2
0.064–0.5
4.4–34
>100 (granules)
Lincosamides Clindamycin 0.032–0.25 <3 (Lysosome) and 8–43.4
(Cytosol)
Oxazolidinones Linezolid 0.5–4 <1 (Lysosome) and 11
(cytosol)
Penicillins Benzylpenicillin
Amoxicillin
0.008–0.125 <1
Glycopeptides Vancomycin 0.25–2 4 7.8 (Cytosol)
Carbapenems Meropenem 0.016–0.5 1–5 (Cytosol)
Cephalosporins Cefazolin
Ceftriaxone
0.125–2
1–8
<1 Low penetration
phagosome
Sulfonamides Sulfamethoxazole 8–128 1.7–3.6 (Phago lysosome)
(cytosol)
DHFR Inhibitors Trimethoprim 0.25–2 3–21 (cytosol)
(microsomal)
Quinolones Ciprofloxacin 0.064–1 2 2–10.9 (cytosol)
Rifamycins Rifampin 0.004–0.032 2.3–9.8 (Phagosome)
Others Fosfomycin 0.25–32 1.8

14. Antimicrobial nanoparticles
• Certain nanoparticles posses potent antibacterial activities and may help to potentiate
small molecule antibiotics.
• The antibacterial activities of aluminium oxide, titanium oxide, zinc oxide, cuprum
oxide, ferum oxide, and silver nitrate nanoparticles against Gram-positive and Gram-
negative pathogens is well documented.
• The cationic charges of titanium and aluminium oxide nanoparticles promote their
adsorption onto bacterial surfaces, destabilizing the membrane, and leading to cellular
leakage.
• Silver nanoparticles produce free radicals that cause lipid peroxidation of the
membrane, resulting in loss of respiratory activities.
• Zinc nanoparticles internalized by bacteria can induce production of ROS and nitric
oxide, resulting in ROS-mediated cell damage.
• Nanoparticles being to large to enter through diffusion enter mammalian cells through
phagocytosis or the pinocytosis pathways.
• Certain nanoparticles such as liposomes, polymeric nanoparticles, solid lipid
nanoparticles and dendrimers can be tailored to display desired charge or composition
for combination with other biomolecules; for example, drugs, antibodies, proteins and
oligonucleotides.
• The nanoparticle surfaces may carry material responsive to a certain stimuli (pH or
temperature) allowing for controllable drug release in a specific place, for example in
the acidified endosome.
• The efficacies of penicillin, gentamicin and tetracycline against intracellular S. aureus,
rifampicin and isoniazid against intracellular M. tuberculosis, streptomycin and
doxycycline against intracellular of Brucella melitensis and rifampicin and
azithromycin against intracellular Chlamydia trachomatis, have all been markedly
improved over the free drug than administered through nano-particlemediated delivery.

15. Nanoparticles as vehicle for antimicrobials
Nanoparticles base (plate-farm material) Antimicrobial delivered
1. Gum acacia (Acasia nilotica), gum jhingan (Lannea coromandelica) Silver nitrate
2. Squanelene Penicillin G
3. Chitosan Tetracycline
4. Polyethylenimine Coating mesoporous silica Rifampicin
Poly d-L-lactide-coglycolide polymer Rifampicin, azithromycin
5. Poly ethylene oxideb-sodium acrylate Streptomycin, doxycycline
(PEO-b-PAA-+Na) and poly sodium acrylate Gentamicin
(PAA-+Na) copolymers
In Bruellosis therapy
Liposomes containing aminoglycosides have been shown to enhance the killing of intra-cellular
Brucella abortus. A 20 mg/L concentration positively-charged SPLV containing 30%
cholesterol and gentamicin completely eradicated the Brucella abortus infection in murine
macrophages. Vitas et al. 1996.
Gentamicin-loaded PLGA 502H microparticles significantly decreased the intracellular Brucella
abortus levels (Prior et al., 2004; Lecaroz et al., 2006).
PEO-b-PtBA diblock copolymer nanoparticles carrying streptomycin and doxycycline have been
used for brucellosis treatment as alternative systems to PLGA copolymers (Seleem et al.
2009).

16. Antibiotic carriers to intra-cytoplasmic sites
• Different improved drug carriers have been developed for treating
intracellular pathogens, including antibiotics loaded into:
– Polymeric drug carriers
– Liposomes
– Niosomes
– Solid Lipid Nanoparticles (SLN)
– Fullerenes
– Dendrimers
– Zeolites
– Erythrocytes
– Ethosomes

17. Name of Herbal antimicrobial B. abortus (36) B. meli-tensis (10)
Tetracycline 83.3 100.0
Doxycycline 58.3 0.0
Streptomycin 86.1 100.0
Gentamicin 91.7 100.0
Cotrimoxazole 44.4 0.0
Azithromycin 41.7 10.0
Chloramphenicol 75.0 100.0
Ciprofloxacin 88.9 100.0
Erythromycin 25.0 0.0
Amoxycillin+ clavulanic acid 63.9 10.0
Amoxycillin 63.9 10.0
Amoxycillin+ sulbactam 69.4 10.0
Ampicillin 19.4 0.0
Aztreonam 50.0 30.0
Cefotaxime* 94.4 100.0
Cefoxitin 75.0 0.0
Ceftazidime 55.6 0.0
Ceftriaxone* 94.4 100.0
Imipenem 100.0 100.0
Meropenem 94.4 100.0
Nitrofurantoin 80.6 90.0
Piperacillin* 85.0 NT
Piperacillin Taztobactam* 90.0 NT
Tigecycline 100.0 100.0
Susceptibility (% sensitive) of Brucella strains to antibiotics (Singh et al., 2019)

18. Management of Brucellosis
In Chinese & Ayurvede (Human)
• Duhuo Jisheng Tang (Giloy), a traditional Chinese herbal medicine used
to treat osteoarthritis , with antibiotics (Sheng FY, 1993).
• Decoctum/ extracts of Amberved (Teucrium polium), barberry, garlic,
Scrophularia deserti, Alhagi and Eucalyptus are used for the treatment
of this disease (Naghdi et al., 2016).
• Maha sudarshan churna, Shadangadi churna, Guduchi satva,
Amritarishta, etc., to manage fever.
• Decoction of guduchi (Giloy), tulsi (Holy basil), sunthi (dried ginger) ,
Black pepper (Piper nigrum), Safron (Crocus sativus) and gud (Jaggery)
in empty stomach.
• To improve appetite and digestion, Kwath of Ginger powder, coriander
seeds and musta in water, few sips every hour.
• Guluchyadi kashayam, Amrutadi guggulu, Amruthotharam kashayam,
Rasnairandadi kwatham and Dashamoola churna to reduce joint pain
and swelling.
• Giloy/ Guduchi (Tinospora cordifolia) to improve immunity and prevent
recurrence. Stimulation of innate immunity can reduce Brucella
burden in the mouse model proven at MSU, 2019.
(http://grantome.com/grant/NIH/R21-AI144496-01)
• Source for more: https://nirogam.com/what-is-brucellosis-and-how-to-treat-it-
with-ayurveda/ & http://www.indianmedicinalplants.info/articles/Brucellosis.html

19. Susceptibility (% sensitive) of Brucella strains to
herbal antimicrobials
Name of Herbal antimicrobial B. abortus (36) B. melit
ensis (10)
Ajowan oil 100.0 100.0
Guggul oil 22.2 0.0
Carvacrol 100.0 100.0
Cinnamon oil 91.7 100.0
Holy basil oil 44.4 0.0
Cinnamaledehyde 100.0 100.0
Lemongrass oil 36.1 80.0
Sandalwood oil 47.2 0.0
Zanthoxylum rhetsa essential oil 13.9 0.0
Agarwood Oil 27.8 0.0
Patchouli (Pogostemon cablin) oil 36.1 10.0
MHARI 0.437 0.555

20. Management of Brucellosis
In Homeopathic medicine
• Nosodes. Melitine, Bacterium melitense Nos, BRUCELLA MELITENSIS 11C,
15C, 30C, 45C, 60C, 75C, 100C, 250C, 500C (For temporary relief of
symptoms related to Brucella infection, 1-10 drops under the tongue, 3 times
a day ) (Barrucand D. Bovine brucellosis; its deep cause; its homeopathic treatment. Homeopath
Fr. 1952 Mar;40(3):174-81.)
• Effective to variable extent in human with reported recoveries in human:
Bryonia, ferrum phos, alfalfa, pulsetilla, ipecac, tuberculinum, sepia, ars
alb, calc carb, aconite (Blaganur AS. 2010. Efficacy of Homeopathic treatment in in case
of brucellosis with serotype changes.
http://52.172.27.147:8080/jspui/bitstream/123456789/2878/1/amaresh%20s%20balaganur.
pdf. ).
• Bacilium 1M, 3 ml weekly; Arsenic iodatum 1 M 3 ml/ day for 6 month
(Dr. Ravi Prkash) in cattle.

21. Brucella phage therapy
• Bogdanov (1938) first succeeded in identifying lysis by a non-specific bacteriophage and
adapting the phage to the lysis of Brucella melitensis.
• The first five strains of Brucella phage active against Brucella abortus were isolated in
1940 from soil and river water.
• Drozevkina, Misnaevskij & Uraleva (1957) and by Drozevkina, Novosel'cev, Uraleva &
Mignaevskij (1960) showed that bacteriophage remains in the blood of the
overwhelming majority of brucellosis patients. At the same time, during the infectious
process a rise in phage titres is noted as the condition of the patients improves.
• Prostetova (1959) showed that Brucella phage is capable of persisting for a long time
both in a healthy guinea pig (up to 30 days) and in a guinea pig infected with brucellosis
(over 45 days).
• Administration of phage to guinea pigs and rabbits suffering from brucellosis led to a
swifter rise in agglutination titres and to an increase in the rate at which animals rid
themselves of brucellae (Drozevkina, 1963).
• Dr. Rawat and his team in IVRI and Saxena et al (2018) at GADVASU proved that phage
therapy has protective and therapeutic efficacy of phage therapy in mice without any
adverse effect on mice health (2014).
• Recently a project is going on in MSU, USA for evaluating Brucella-specific phage with
immune boosters for brucellosis. The phages will be combined with innate
immunostimulation and encapsulated into
liposomes. (http://grantome.com/grant/NIH/R21-AI144496-01)

22. Critical gaps
• Though knowledge exists about the
antimicrobials and alternative therapies to
cure Brucellosis, there is no applied research
in the field or validated efficacy studies in
animals.
• Lack of any clinical therapy for the cure of
brucellosis in Dairy animals is a big hurdle in
the control and containment of Brucellosis in
livestock.

23. Objective: To evaluate therapeutic interventions
for Brucellosis in cattle and buffaloes.
Long-term objective: To ensure preservation
and propagation of precious cattle and buffalo
germ-plasm, often more susceptible, and
discarded due to brucellosis.
Practical utility : The Disease is a prioritized
disease for control at the National level so the
development of any potential therapy will
boost the disease control program.

24. Technical Program
• Screening of IVRI, Dairy animals for Brucellosis and
recruitment into the study after due approval. Using
ELISA, RBPT and STAT
• All positive animals will be screened for excretion of
Brucella in milk, vaginal secretions/ excretions and, and
from postmortem cases from the spleen, uterus and
inguinal and mammary lymph nodes.
• Grouping of animals after randomization for
– Immunotherapy
– Antibiotic Therapy
– Homeopathic Therapy
– Phage Therapy
– Herbal antimicrobial / nano-medicine therapy

25. Immunotherapy & Phage Therapy
In January 2019, Montana State University granted a project on use of
Brucella-specific phages, in combination with innate immune
stimulation, as a novel countermeasure for brucellosis
(1R21AI144496-01). However, we plan for:
A. Retesting of Saxena & Raj, 2018 Protocol: Vaccination with
phage lysates of RB51 and S19 strains of B. Abortus, single
subcutaneous dose of 2 ml of cocktail inactivated lysate is claimed
curative in cattle.
B. S-19/ RB-51 Phage-lysate without inactivation but filter
sterilization. With immune boosters as Sat Giloy (Tinospora
cordifolia).
C. Brucela specific phages encapsulated in liposoml nanovesicles
and will be used with Sat Giloy (Tinospora cordifolia).
Each Trial will be conducted in 6-18 animals depending on
availability of positive animals. All animals will be monitored
for Brucella antibodies fortnightly.

26. Antibiotic Therapy
• A. Streptomycine+ Doxicycline for 3 months
(used in humans)?? With immune boosters as
Sat Giloy (Tinospora cordifolia).
• B. Streptomycin+ Tetracycline (proposed for
animals) for 3 months??? With immune
boosters as Sat Giloy (Tinospora cordifolia).
Each Trial will be conducted in 6-18 animals
depending on availability of positive animals. All
animals will be monitored for Brucella
antibodies fortnightly for the whole duration of
the project.

27. Homeopathic Therapy
• Preparation of Nosodes from B. abortus and B.
melitensis.
• Use of Nosodes with other drugs such as Ars
iodatum and more yet to be decided according to
underlying signs and body structure/ physiology
and behaviours of patients such as Bryonia,
ferrum phos, alfalfa, pulsetilla, ipecac, sepia, ars
alb, calc carb, aconite etc.
• Trial will be conducted in 6-18 animals
depending on the availability of positive
animals. All animals will be monitored for
Brucella antibodies fortnightly for the whole
duration of the project.

28. Herbal nano-therapy
• Synthesis of Nanoparticles loaded with ajowan/
cinnamon oil with and without Green synthesized
Silver nanoparticles.
• In-vitro evaluation of nanoparticles and their
combination on Brucella isolates, for their penetration
and drug delivery in macrophages and intracellular
Brucella killing ability.
• The best combination trial for safety and efficacy in 6-
18 Brucellosis-positive animals. All animals will be
monitored for Brucella antibodies fortnightly for the
whole duration of the project.

29. References: Sources of Information
1. Bongers et al. 2019. Intracellular Penetration and Effects of Antibiotics on Staphylococcus
aureus Inside Human Neutrophils: A Comprehensive Review. Antibiotics (Basel). 2019 May
4;8(2). pii: E54. doi: 10.3390/antibiotics8020054.
2. Sigh BR. 2019. Antimicrobial Therapy for Intracellular Bacterial Infections.
DOI: 10.13140/RG.2.2.15572.35206
3. Kamaruzzaman et al. 2017. Targeting the hard to reach: challenges and novel strategies in the
treatment of intracellular bacterial infections. Br J Pharmacol. 174(14):2225–2236.
doi:10.1111/bph.13664.
4. Chifiriuc et al. 2016. Antibiotic Drug Delivery Systems for the Intracellular Targeting of Bacterial
Pathogens. https://www.intechopen.com/chapter/pdf-download/49265.
5. Prajapati A-----and Rawat M (2014) Therapeutic efficacy of Brucella phage against Brucella
abortus in mice model, Veterinary World 7(1): 34-37.
6. Kumar S, Singh BR . 2013. An Overview on Mechanisms and Emergence of Antimicrobials
Drug Resistance. Advances in Animal and Veterinary Sciences. 1 (2S): 7 – 14.
7. Toti et al. 2011. Targeted delivery of antibiotics to intracellular chlamydial infections using PLGA
nanoparticles. Biomaterials. 2011;32(27):6606–6613. doi:10.1016/j.biomaterials.
8. Gamazo et al. 2010. Drug delivery systems for potential treatment of intracellular bacterial
infections. https://pdfs.semanticscholar.org/6d89/a16a8605a5a68e34352821f41be78f2ae1b8.pdf.
9. Maurin M, Raoult D. 2001. Use of aminoglycosides in treatment of infections due to intracellular
bacteria. Antimicrob Agents Chemother. 45(11):2977-86.
10. Maurine et al. 2000. Bactericidal activities of antibiotics against intracellular Francisella
tularensis. Antimicrob Agents Chemother. 44(12):3428-31.

30. A cross-sectional survey of Holy Cow
Infectious Problems in Gaushalas
Gaushalas are protective shelters for stray cows in India
Duration: September 2020 to August 2022
PI: Bhoj R Singh

31. The Holy cow (Gau Mata) has
reached now in shelter homes
• What a son are you?
– You love your mother and often claim yourself ‘Matrubhakt”.
– However, your mother is living in a shelter home.
– You have made rearers & nurtures of Holy cows to abandon your
mother.
– You made your Holy mother to as unholy as possible, forced her to
steal in the night, on run in say time & eat wastes & excreta.
– Thousands of mothers are now lame and crippled suffering from
incurable ailments.
– Mother is dying of slow death in a shelter, abandoned on roads and
jungles.
– Her body is bleeding from cuts given by barbed wires.
– Mother is crying but you are deaf and dumb due to the hollow sound
of your false claims for the protection of GauMata.

32. Problems of Cow-shelters
Sharma et al., 2020.
• Too many animals in too small a space.
• Poor nutrition, only Rs. 30/ to feed a cow daily.
• Too many injured but a few doctors.
• Too many known and unknown diseases.
• Exploitation of Gaumata, Gauvats and
Gausewaks.
• Nexus of management with Killers of Holy Cow.
• Poor hygiene.

33. Health Problems in Gaushalas
• Very few or No Systematic studies!!
• The majority of the cattle sheltered in gaushalas are likely to be
immunocompromised, with infectious disease-causing agents
like Listeria spp., Streptococcus spp., Staphylococcus spp.,
and Corynebacterium spp., Brucella spp., Mycobacterium spp.
predominating due to the unhygienic environment (Sharma et al., 2020).
• 15.5% prevalence of brucellosis in gaushala cattle and 4.5% in the workers
employed in the gaushalas (Singh et al., 2004)
• The prevalence of tick infestation in Gaushalas and unorganized dairy
farms has been reported as 45%, but only 4% in the organized sector.
(Singh et al., 2018, Surabhi et al., 2018).
• Many shelters reported outbreaks of FMD in the last five years, which
might be due to poor biosecurity, as the majority of the shelters
vaccinated their animals against the disease. FMD is endemic in India,
spreading by direct contact with infected animals, fomites of workers,
fodder and feeding utensils (Kandel et al., 2018).
• The median mortality rate of 13.8% (Thomsen & Houe, 2006).
• 95% of stray cattle had gastrointestinal disorders following ingestion of
plastic bags and other foreign bodies(Singh, 2005).
• Separation of calves from their mothers in 40% of the shelters is also a
welfare concern (Sharma et al., 2020).

34. Important Infectious Problems
• Wounds and abscesses (Actinomycosis, Actinobacillosis,
Streptococcus spp., Staphylococcus spp., and Corynebacterium spp.
Infections)
• Brucellosis
• Tuberculosis
• FMD
• LSD
• HS
• BQ
• Anthrax
• IBD
• IBR

35. Objectives
• Determining quantitative estimates of health
problems of selected Gaushalas permitting
sampling in Northern India.
• Long-term objective: Protection of Holy Cow
in India

36. Technical program
• Collection of data on health problems in cattle in
Gaushalas and Gaushala caretakers.
• Random sampling from Gaushala cattle and
caretakers for faecal parasitic load, blood
serology (biochemical analysis, antibody assays
and PCR assays for selected pathogens) & urine
(for routine and microbiological examination
using conventional molecular biology tools).
• Data Analysis and Report writing.
• Development of recommendations for holy cow
health

37. Explorative study on Epidemiological
determinants associated with drastic
reduction in Milk Production of Dairy
Animals with reference to
communicable diseases
Duration: September 2022-August 2024
PI: Bhoj R Singh
Collaborators: Dr. DK Sinha (Epidemiology), Dr.
VK Chaturvedi (B&M), Dr. Vinodh Kumar OR
(Epidemiology), A scientist from LPM

38. Problem identification
• In previous years at our Institutional Dairy
Farm several incidences of sudden drop in
milk production have occurred with no or little
explanation and understanding of the causes.

39. Common causes of drastic milk
reduction
• Managerial (Nutritional, housing, crowding etc.)
• Environmental (heat/ cold stress)
• Physiological (acetonemia or ruminal acidosis, late
pregnancy, breeding heat, displaced abomasum)
• Pathological: Acute infection (Viral, Bacterial,
parasitic, fungal). A sudden onset of pneumonia,
diarrhoea caused by BVD, Salmonella, Winter
dysentery, FMD, HS, Leptospirosis, subclinical or clinical
mastitis, Vitamin B12 deficiency, mycotoxicosis (Ishler,
2016), infectious bovine rhinotracheitis virus (IBR) &
Influenza A virus infection (Gunning, 2002), acute
theileriosis, babesiosis, anaplasmosis and others as
anthelmintic treatment and vaccination (Ravinet et al.,
2016) can also cause sudden drops in milk yield.

40. Sudden drop in Milk production
• ≥10% drop in milk production in a day is
considered alarming (https://extension.psu.edu)

41. Objectives
• To determine the factors associated with a
sudden drop in milk production at ICAR-IVRI
dairy farm using Retrospective and
prospective analysis to minimize the losses.
• Long-term objective: Increasing productivity
of Dairy animals

42. Technical program
• Retrieval of milk production data for the last ten years and
for two coming years for individual dairy animals and
cumulative to identify the incidences of sudden drop in
milk production at individual animal level, and farm level.
• Retrieval of nutritional, managemental, environmental
(temperature & humidity), deworming, vaccination and
disease data for the last ten years and for two coming years
a week before and a week after all incidences of sudden
milk drops.
• Data entry for suitable analysis (Relative risk and
attributable risk).
• Interpretation
• Formulation of recommendations for mitigating the
problems of sudden milk drop.

43. Thanking you for reading the rejected
research proposals