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Concepts of bypass protein feeding in ruminants

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Nilufar Haque Shahed Hossain

West Bengal University of Animal and Fishery Sciences American International University-Bangladesh
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 VOL.5 ISSUE 11 MARCH 2012

Editor : B.V. SHIV SHANKAR

Sub Editor : R. SHAILESH NAATH

CONTENTS

1. From the Editor's Desk 3

2. Antimicrobial Dosing Regimens: a Dynamic... 4-8

- Dr. Vijaykumar.M

3. Concepts of Bypass Protein Feeding in... 9-13

- Dr. Nilufar Haque

4. A Few Nutritional Updates for Feeding... 14-17

- Dr Trishna B. Kayastha

5. Common Diseases of Livestock Caused… 18-24

- Dr. Phaniraja.K.L

6. Heat Stress Takes Toll on Dairy Animal 29-31

- Dr.Arindam Chatterjee

7. Livestock Improvement Strategies in... 32-34

- Dr. Dibyendu Chakraborty

8. Importance of Biotechnology in Animal... 35-36

- Dr. Dharmendra Vyas

9. Modifications of Polymerase Chain... 37-44

- Dr. Arunkumar Patel

10. Mycotoxin: A Threat to Human and... 45-48

- Dr. Mahipal Choubey

11. Impact of nanotechnology in veterinary... 49-50

INDEX OF ADVERTISEMENTS - Dr. S. Ganguly
1. B.V. Bio-Corp. Pvt. Ltd. Title Cover I
2. DSM Nutritional Products India Pvt. Ltd. Inside Colour 25
12. Press Release 51-52
3. Jefo Title Cover II
4. Kemin Industries South Asia Pvt Ltd. Inside Colour 28
5. Polyglov 2
6. Trow Nutrition India Pvt. Ltd. Title Cover III
7. Vetoquinol India Animal Health Pvt. Ltd. Title Cover IV

B.V. Shiv Shankar - Managing Partner

B. Kishore Kumar - Media Executive
B. Shailajaa - Circulation Manager
Sathyendranath - Marketing Manager
B. L.Narasimham - Regional Representative
K. Raghuramaraju - Publication Consultant (09440231211)

Printed, Published and Owned by B.V. Shiv Shankar, Printed at Venu Graphics, 2-1-392/1/3/8, Opp. Fever Hospital, Nallakunta, Hyderabad - 500 044. India.
Published at 2-1-444/16, 1st Floor, O.U.Road, Nallakunta,Hyd-44. Editor: B.V. Shiv Shankar.

LIVESTOCK LINE, MARCH 2012 1

LIVESTOCK LINE, MARCH 2012 2
 From the Editor's Desk....…..

OPERATION FLOOD
The National Dairy Development Board’s (NDDB) Annual Report for 2010-11 has conveyed
that India continued to be the largest milk producing nation in 2010-11. The country’s
estimated milk production for 2010-11 is 121 million tonnes, close to 17 per cent of world
milk production.

This was possible with government schemes like Mahatma Gandhi National Rural
Employment Generation Agency (MGNREGA) which aims at rural employment generation,
milk production etc. The NDDB on its side offers technical assistance for balanced diet,
increase in milk production etc and devised a plan known as National Dairy Plan (NDP) with
a good span of 15 years hence.

The National Dairy Plan aims at contributing to increasing milk production by increasing
productivity in existing dairy animals through a focused and scientific process for breeding
and feeding.

Production of high genetic merit bulls,

Production of disease free quality semen,

Extension and demonstrations for fodder development,

Interventions to strengthen village based milk procurement systems,

Augmenting systems in the villages for procurement of milk in a fair and transparent
manner,

Project learning and monitoring and capacity building and training.
The project is proposed to be carried out by State Cooperative Dairy Federations; District
Cooperative Milk Producers Unions; Producer Companies and State Livestock development
Boards that meet the criteria for each activity.
Existing farmers and new entrepreneurs may take advantage of this programme and
augment their personal wealth as well as nation’s wealth.

- Editor

LIVESTOCK LINE, MARCH 2012 3

 Antimicrobial Dosing Regimens: a Dynamic Challenge
Vijaykumar.M1, U.Sunilchandra2 and Meera V.C3 and Shrikant kulkarni 4
Department of Pharmacology and Toxicology
1
Department of Veterinary Physiology, KVAFSU, Veterinary College, Bidar.Karnataka.
U
Dosing regimens for antimicrobials exemplify the
integration of pharmacokinetics (what the body does
to the drug) and pharmacodynamics (what the drug
does to the body). For antimicrobial therapy, the
“body” is the microbe. Integration is based upon what
is needed to achieve the pharmacodynamic
response—in this case, the minimum inhibitory
concentration of the drug of interest for the infecting
microbe—and comparing it with what will be achieved
at the chosen dose. For this discussion, we will build
a dose around a desired pharmacodynamic index
(PDI). If the MIC of the infecting is not known, then
the MIC 90 is a reasonable surrogate. The MIC90 is
the MIC at or below which 90% of the isolates is an
sample population of the organism is inhibited.
1.Relationship Between MIC, Plasma and
Tissue Drug Concentrations: The parameters that
are most predictive of antimicrobial efficacy and lack
of resistance are the ratio of Cmax / MIC, the area
under the inhibitory curve (AUC/MIC); and the
percent time that PDC are above the MIC [T> MIC].
Based on these relationships, two generally
categories of drugs have been described. Exceeding
the efficacy targets decreases resistance. Dosing
regimens can be designed based on population
statistics, using MIC 90 (e.g., packaged inserts) as
a surrogate indicator of what is needed, or using
MIC data from a culture report. The design of the
dosing regimen depends upon the drug and its
relationship between plasma drug concentrations,
the MIC of the infecting organisms and whether or
not the drug has a substantial post antibiotic effect
(PAE). The PAE refers to the continued inhibition of
microbial growth after a short exposure of the
organisms to the drug. The impact is particularly
profound for concentration-dependent drugs, and
allows some drugs to be administered at long dosing
intervals. The PAE may be absent for some
organisms or some patients (e.g., some
immunocompromised patients). In general, the PAE
of concentration-dependent drugs is increased as
the Cmax/MIC increases.
1,2,3
Assistant Professor,Department of Pharmacology and Toxicology
4
Assistant Professor,Department of Veterinary Physiology
LIVESTOCK LINE, MARCH 2012
2.Time versus Concentration Dependent
Drugs: The relationship between MIC and the
magnitude and time course of PDC allows drugs to
be categorized as to either concentration-dependent
(sometimes referred to as dose dependent) or time-
dependent; these definitions are supported by
primarily by in vitro but also in vivo studies.
Concentration dependent drugs, best represented
by the fluoroquinolones and aminoglycosides, are
characterized by efficacy best predicted by the
magnitude of plasma drug concentration (C max)
compared to the MIC of the infecting organism. For
such drugs, the magnitude of the ratio generally
should be 10 to 12, but ideally is higher for more
difficult infections (e.g., Pseudomonas aeruginosa,
or infections caused by multiple organisms. The
duration that PDC is above the MIC is not as
important; in fact, efficacy may be enhanced by a
drug-free period (i.e., a long interval between doses).
For concentration dependent drugs, a dose that is
too low is particularly detrimental. As such,
concentration-dependent drugs generally can be
administered at longer intervals, i.e., once a day.
Package inserts can be used to demonstrate the
design of a dose for a FQ. As such, theoretically the
low dose would be appropriate for treatment of both.
For Staph intermedius, with an MIC 90 of 0.25 mcg/
ml, 2.5 mcg/ml is the target. The higher dose of 5.5
mg/kg would be more prudent. For organisms with
an MIC of 0.5 mcg/ml, the target of 5 mcg/ml could
not be reached at 5.5 mg/kg. However, for the
fluorinated quinolones (FQ), efficacy also is
predicted in vitro by AUC/MIC: a ratio of < 60 renders
the drugs bacteriostatic, whereas > 125 results in
(slow) killing but also decreases the risk of resistance
and > 250 causing more rapid bacterial killing. Thus,
resistance might be less likely to develop for FQ
characterized by longer half-lives (or for ENR, by
the production of an active metabolite). Twice daily
administration of an FQ might be indicated for
organisms already characterized by low level
resistance (see MPC below); however, the once daily
dose should be given twice daily in such situations.
4
 The use of a second drug in combination with the MIC 90 for Staphylococcus intermedius is 2 mcg/ml;
FQ might also be considered for isolates whose MIC a dose of 25 mg/kg achieves 15 mcg/ml. The amount
are sufficiently high that a Cmax/MIC >10 is difficult of time that can lapse can be calculated as follows:
to achieve. the Cmax/MIC (15/2)= 7.5. This equates to essentially
8, or 3 half-lives (2*2*2). Thus, 4 hours can lapse
3. Time Dependent Drugs : In contrast to
during T>MIC; a dosing interval of 8 hrs is indicated.
concentration dependent drugs, efficacy of time-
(To check: 15 mcg/ml > 7.5 > 3.85 > 1.9 mcg/ml = 3
dependent drugs (e.g., â-lactams) is enhanced if
half-lives). For Staphylococcus aureus, the MIC90
PDC remain above the MIC for the majority (50 to
is 8 mcg/ml; the Cmax/MIC = 15/8 = essentially 2.
70%) of the dosing interval; efficacy is best predicted
One half-life can lapse during T>MIC; the dosing
by percent time that PDC are above the MIC [T>
interval can be 2 half-lives, or 3 hours long.
MIC]. For such drugs, simply achieving the MIC
Increasing the dose to treat St. aureus at a
(Cmax/MIC =1) is insufficient because PDC (and
convenient dosing interval is not practical. For E coli,
certainly tissue concentrations) fall below the MIC
with an MIC 90 of 16 mcg/ml, not even one half-life
immediately. With time-dependent drugs, generally
can lapse. Cephalexin should not be used to treat E
a Cmax/MIC of 4 is a good starting point because it
coli. In general, for time dependent drugs, especially
assures 2 half-lives will lapse before T=MIC. Two
if the half-life is short, adding an additional dose is
more half-lives can then be added to the dosing
more cost effective than increasing the dose. This
interval before the next dose must be given if T>MIC
is in contrast for time dependent drugs that have a
50%. However, while this sounds like a long time, for
long half-life. For example, once daily dosing may
amoxicillin and cephalexin, with a half-life of about
be appropriate for cefpodoxime, depending on the
1.5 hr, the dosing interval can only be 6 hrs if Cmax/
organism. According to the package insert, the MIC
MIC = 4. For example, the MIC 90 for Staph
90 for both Staph intermedius and E coli is 0.5 mcg/
intermedius and amoxicillin-clavulanic acid is <
ml. The Cmax at 10 mg/kg achieves 16 mcg/ml. The
0.5 mcg/ml. Cmax of 5.5 mcg/ml will be achieved at
number of half-lives that can lapse is 16/0.5 = 32 =
the labeled dose of amoxicillin-clavulanic. The
5 half-lives (2X2X2X2X2), or 16 > 8 > 4 > 2 > 1 > 0.5.
duration of the dosing interval with this dose depends
The half-life of cefpodoxime is 4.5 hrs, thus the
on the number of half-lives that can lapse as drug
dosing interval can be 25 hr X 2, or (theoretically)
concentrations decline to the MIC. In one half-life,
every 2 days. However, the variability in drug
plasma drug concentrations will be 2.75 mcg/ml; in
concentrations is marked, and prudence suggests
two half-lives, 1.35 and in three half-lives, 0.65, which
that a 24 hour dosing regimen, as is indicated on
is just above the target. The half-life of amoxicillin is
the label, is appropriate. For Staph aureus, the MIC
at best 1.5 hrs resulting in 4.5 hrs of T>MIC. The
90 is 2 mcg/ml. Three half-lives can lapse during
dosing interval can be twice this long. Thus, the next
T>MIC; 6 half-lives or essentially a day can lapse
dose should be administered at 9 hrs (8 hr). To reach
before the next dose. However, because this
a 12 hr dosing interval, 3 more hours, or two half-
facilitates efficacy, but not necessarily avoids
lives are needed. T>MIC is needed for one more
resistance, and because these calculations assume
half-life; thus, the dose needs to be doubled. Thus,
all drug in plasma makes it to the site of infection, a
to treat an organism with an MIC of 0.5 mcg/ml with
12 hour dosing interval might be more prudent.
amoxicillin-clavulanic acid, a dose of 13.5 mg/kg
every 8 hrs, or 27 mg/kg every 12 hrs must be given. For cefovecin, with a 133 hr half-life (due to protein
The dose would need to be further modified for drug, binding which slowly releases the drug), for each 2X
microbial and host factors. Staphylococcus Cmax/MIC, 6 days can lapse (assuming time
intermedius is characterized by a low MIC; if we dependency is valid for periods beyond 24 hrs).
repeated the process for Staph. aureus, its MIC 90 However, the Cmax must be based on unbound, not
is 4 mcg/ml. At 13.5 mg/kg, drug concentrations will bound drug. The Cmax of unbound drug in dogs
reach the MIC before one half-life lapses. Even for approximates 4.0 mcg/ml. The MIC 90 for Staph
E. coli, with an MIC90 for amoxicillin-clavulanic acid intermedius is 0.25 mcg/ml; approximately 4 half-lives
at 1 mcg/ml, a dose of 26 mg/kg every 8 hrs is the can lapse during T>MIC; approximately 8 half-lives
minimum that should be considered. The process (40 days) can lapse before the next dose is given.
can be repeated for cephalexin, with a half-life of However, if the target organism is Staph aureus, with
1.3 hr. At 25 mg/kg PO, 15 mcg/ml is achieved. The

LIVESTOCK LINE, MARCH 2012 5

 an MIC of 2 mcg/ml, T>MIC for only one half-life and variable; we will us an average of 4 hours. Thus, if
dosing should occur (if indicated) in one week or the dog could tolerate it (unlikely), a dose of 80 mg/
less. Constant rate infusion or slow release products kg every 8 hours would be indicated. However, this
might be ideal for time dependent drugs with short- would only result in bacteriostatic concentrations.
half-lives in the critical patient. Slow release products The combination with rifampin would at least increase
might be considered for time dependent drugs; the changes of therapeutic success.
however, the dose must be designed to assure that
The previous demonstrations have been based on
the MIC is achieved for the older slow release
the assumption that all drug in plasma makes it to
products because MIC have changed through the
the site of infection. However, a variety of host, drug
years. Azithromycin is another example of a drug with
and microbial factors should cause the dosing
a very long half-life (72 hr) because of tissue
regimen to be modified even further.
distribution and accumulation. Although the drug can
be administered at 2 day intervals. depending on 4.Host Factors: The impact of host response to
the target MIC, note that the drug may not reach infection can be profound. Problems contributing to
steady-state concentrations for 6 to 14 days. Indeed, therapeutic failure include immunocompromise
care must be taken to remember that the maximum (design a dosing regimen that will assure bactericidal
effect of any drug with a long half-life will not be concentrations of the chosen drug reach the site of
achieved for 3 to 5 half-lives and a loading dose infection), inflammatory response (debride or
might be indicated for such drugs. Finally, some time otherwise appropriate clean/drain accessible
dependent drugs have a very long half-life. infections, select a drug that distributes into tissues
well and ideally accumulates in phagocytes and
Based in the Pseudomonas aeruginosa, the dose
increase the dose appropriately). Interpretation of
for amikacin should be sufficient to achieve Cmax/
C&S is based on the assumption that the MIC should
MIC = 10 or 10 X 16 mcg/ml = 160 mcg/ml. Table 1
be achieved in plasma. Basing MIC interpretation
indicates that 22 mg/kg achieved 64 mcg/ml in the
on plasma drug concentrations (PDC) might result
blood stream. One could calculated the dose needed
in over or under estimation of drug efficacy. For
to achieve 160 mcg/ml based on either a proportion
tissues which concentrate the drug (or if the drug
of : (160 mcg/ml)/(64 mcg/ml) = 2.5 X 22 mg/kg = 55
can be applied topically), and for drugs which can
mg/kg; or one could calculate the dose based on
be concentrated by phagocytes and thus transported
target (160 mcg/ml) X Vd = 37 mg/kg. Either
to the site of infection, concentrations may markedly
calculation is likely to result in the same conclusion:
exceed PDC, resulting in underestimation of efficacy
the drug may not be safe at this high dose, even
for several reasons. Much of the data for water
with once daily dosing, and the addition of a second
soluble drugs (volume of distribution [Vd] generally
drug is indicated. For enrofloxacin, the target is 1
< 0.3 L/kg) suggests antimicrobial concentrations
mcg/ml * 10 = 10 mcg/ml. At 20 mg/kg, enrofloxacin
may be 30% or less of PDC in some tissues,
achieves 4 mcg/ml and its active metabolite
particularly those characterized as sanctuaries, i.e.,
ciprofloxacin, achieves 2.9 mcg/ml for a total
non-fenestrated capillaries. In humans,
bioactivity of approximately 7 mcg/ml. Interestingly,
recommended doses of beta-lactams drugs (water
despite an “I” designation, enrofloxacin comes closer
soluble) are increased 5 to 10 fold when treating
to achieving the target Cmax/MIC of 10 (7 is
infections of the central nervous system. Even tissues
achieved). For enrofloxacin, a second 20 mg/kg dose
traditionally considered “well perfused” might be of
could be added. However, the combination of
concern. For example, drugs do not penetrate
enrofloxacin and amikacin would be a wiser choice.
bronchial secretions well, despite the fact that the
For the MRSA, note that chloramphenicol, despite
lungs are well perfused. Amoxicillin is often used to
an “S” designation, requires an MIC of 8 mcg/ml.
treat respiratory tract infections. Yet, only 30% of
The dosing table indicates that 55 mg/kg PO will
the amoxicillin that is in plasma is distributed to
achieve a Cmax of 10 mcg/ml. Thus, not even one
bronchial secretions. Theoretically, one must dose
half-life can lapse before T>MIC is reached. A dose
amoxicillin 3X the recommended dose to achieve
of 80 mg/kg would achieve approximately 16 mcg/
targeted PDC in bronchial secretions. Most water
ml, which would allow one half-live of T>MIC, or a 2
soluble drugs (beta-lactams and aminoglycosides)
half-life dosing interval. The reported half-life is
reach only 20 to 25% of PDC in bronchial secretions

LIVESTOCK LINE, MARCH 2012 6

 whereas over 50% of lipid soluble drugs reach
bronchial secretions. Dosing adjustments also are
necessary for those infections that are intracellular
or complicated by host response to infection. In the
presence of marked inflammation, use of a drug that
accumulates in phagocytes (e.g., FQs, macrolides,
lincosamides) is likely to increase distribution of the
drug to the site.
5.Microbial Factors: Materials released from
microbes facilitate invasion, impair cellular
phagocytosis, and damage host tissues. Most
staphylococci associated with canine pyoderma
produce “slime,” a material that facilitates bacterial
adhesion to cells. Soluble mediators released by
organisms (hemolysin, epidermolytic toxin,
leukocidin) may damage host tissues or alter host
response. Staphylococcal organisms contain protein
A, which impairs antibody response, activates
complement, and causes chemotaxis. Nocardia
stimulates the formation of calcium-containing “sulfur
granules” that impair drug penetration to the
organisms. Pseudomonas and other gram-negative
organisms produce a glycocalix, or biofilm, that
protects the organism. Biofilms are microcolonies of
pathogenic and host microbes embedded in a
polysaccharide matrix (“slime” or “glycocalyx”)
produced by the bacteria; dental plaque is the
prototypic example. Normal microflora of the skin or
mucous membranes in the biofilm are lost with
shedding of the skin surface or by the excretion of
mucus; new cells and mucus are rapidly colonized
by biofilm forming bacteria. Translocation of the
normal microflora to otherwise sterile tissues (which
can be facilitated by the presence of foreign bodies)
may lead to acute infections (again, associated with
biofilm) and accompanying inflammatory response.
Persistent, chronic bacterial infections may reflect
biofilm producing bacteria; persistent inflammation
associated with immune complexes contributes to
clinical signs. Unfortunately, bacteria growing in
biofilms more easily resist antimicrobial killing and
immune defenses of the host. In addition to
debridement or other methods of cleansing should
facilitate antimicrobial penetration; dose modification
(increase) may be indicated to compensate for
debris. Attention to PDC is important not only for
efficacy, but also in order to reduce the risk of
resistance. For drugs in which resistance emerges
as a result of point mutations, dosing regimens
should be designed to target the MPC. The mutant
LIVESTOCK LINE, MARCH 2012
prevention concentration (MPC) is defined as the
highest MIC identified in a population (> 107) infecting
the patient. The MPC, rather than the MIC, should
be the targeted concentration of drug at the site of
infection if resistance is to be avoided. Unfortunately,
determining the MPC of an isolate cultured from a
patient requires culture techniques based on > 107
organisms, which currently is not possible.
6. Drug Factors : In addition to drug characteristics
previously addressed (e.g., concentration versus
time dependent, static versus cidal, drug
distribution), pharmaceutical manufacturers have
been able to manipulate antimicrobial drugs in a
variety of ways such that efficacy and thus bacterial
killing is enhanced such that resistance might be
reduced. For example, efficacy has been decreased
by synthesizing smaller molecules that can penetrate
smaller porins (e.g., the extended spectrum
penicillins ticarcillin and piperacillin); “protecting” the
antibiotic (e.g., with clavulanic acid, which “draws”
the attention of the â-lactamase away from the
penicillin); modifying the compound so that it is more
difficult to destroy (e.g., amikacin, which is a larger
and more difficult to reach molecule than gentamicin);
and developing lipid-soluble compounds that are
more able to achieve effective concentrations at the
site of infection (e.g., doxycycline compared with
other tetracyclines). However, with each innovative
approach to reducing resistance, microbes are able
to circumvent the drug in a disconcertingly short time.
The use of pro-biotics or pre-biotics to minimize
emergence of resistance in the gastrointestinal tract
is controversial and requires additional scientific
evidence.
7.Duration of Dosing : Increasingly, in an effort to
reduce antimicrobial resistance, the duration of
dosing in human medicine is being limited. Durations
of 5 days or less are recommended for non-
complicated infections. High and/or frequent dosing
is intended to result in rapid kill. For slow growing
organisms, or infections complicated by poor local
immunity or prolonged healing, longer durations are
indicated. Note that pulse dosing at high, frequent
doses intermittently might be preferred to long term
dosing at lower doses. The former should be
approached such that mutants are killed. In such
cases, recurrent infection might be caused by
organisms that are not resistant.
References:
7
 1. Papich, M. G. Tissue concentrations of
antimicrobials: the site of action. Problems in
Veterinary Medicine 1990; 2: 312.
2.Ling, G. V. Therapeutic strategies involving
antimicrobial treatment of the canine urinary tract.
Journal of the American Veterinary Medical
Association 1984; 185: 1162.
3.Klausner, J. S. Management of canine bacterial
prostatitis. Journal of the American Veterinary
Medical Association 1983; 182: 292.
4. Bemis, D. A., Appel, M. J. G. Aerosol, parenteral
and oral antibiotic treatment of Bordetella
bronchiseptica infection in dogs. Journal of the
American Veterinary Medical Association 1977; 170:
1082.
5.Pennington, J. E., Reynolds, H. Y. Concentration
of gentamicin and carbenicillin in bronchial
secretions. Journal of Infectious Diseases 1973; 128:
63.
6.Hall, B. B., Fitzgerald, R. H., Kelly, P. J., Washington
J. A. Pharmacokinetics of penicillin in canine
osteomyelitic bone. Orthopaedic Transactions 1980;
4: 175.
7.Wiggins, C. E., Nelson, C. L., Clarke, R.,
Thompson, C. H. Concentration of antibiotics in
normal bone after intravenous injection. Journal of
Bone and Joint Surgery 1978; 60A: 93.
8.Johnson, K. A., Watson, A. D. J., Page, R. L.
Skeletal diseases. In: Ettinger, S. J., Feldman, E. C.
eds. Textbook of Veterinary Internal Medicine.
Philadelphia, W. B. Saunders Co., 1995: 2077.
9.Dwozack, D. L. Emergence of resistance in Gram-
negative bacteria: a risk of broad-spectrum beta-
lactam use. Drug Intelligence and Clinical Pharmacy
1986; 20: 562.
10.Saunders, W. E., Saunders, C. C. Inducible b-
lactamases: clinical and epidemiologica1 implications
for use of newer cephalosporins. Reviews in
Infectious Disease 1988; 10: 830.
11.Marcellin-Little, D. J., Papich, M. G., Richardson,
D. C., DeYoung, D. J. Pharmacokinetic model for
cefazolin distribution during total hip arthroplasty in
dogs. American Journal of Veterinary Research
1966; 57: 720. d its relationship to pyogenic vertebral
osteomyelitis. Journal of Bone Joint and Surgery
1959; 41B: 797-809.


LIVESTOCK LINE, MARCH 2012 8
 Concepts of Bypass Protein Feeding in Ruminants
Nilufar Haque and Sk. Asraf Hossain, National Dairy Research Institute, Karnal, Haryana-132001
Email ID of corresponding author: haquenilufar@gmail.com
U
In the developing countries of Asia and Africa, the
feed inadequacy is the major impediment coming
in the way of development of livestock sector. The
problem is really acute in India, where the bovine
population is the largest (185.2 million cattle and
97.9 million buffaloes according to 17th Livestock
Census, 2003), which is increasing @ 1% annually.
Thus, the constant increase in bovine population in
India dilutes any effort made in increasing the feed
supply to these animals, through non-conventional
feed resources. However, there is also an alternative
way of increasing the nutrient supply to bovines in
these countries, and that is, by modifying the feeds
and the feeding conditions, and also by
manipulating the digestive tract, or through better
feeding management. Such an approach can result
in increasing the feed conversion efficiency of feeds
within the animal system.
Times when AA balance is critical:
1. When attempting to reduce the amount of protein
fed, thereby reducing the cost of the diet and
increasing the space in the diet for higher energy
feeds. For the high producing dairy cow, energy is
usually more in deficit than AA. Lowering protein in
the diet will also reduce the spilling of nitrogen into
the environment and lower the threat of regulation.
This becomes an extra bonus and for herds under
environmental regulation may be a primary use of
AA balance.
2. For fresh cows, both to minimize body condition
loss as well as increase milk production.
3. To increase milk protein (Patton, 2009).
Bypass protein supplement
On account of shortage and to exploit milk
production of dairy animals, limited feed ingredients
available in India should be utilized efficiently with
value addition. The total annual availability of protein
meals in India is approximately 19-20 MMT, against
a requirement of about 30-35 MMT. Out of 20 MMT
protein meals produced in the country,
approximately 4-5 MMT are exported, which further
LIVESTOCK LINE, MARCH 2012
increases the gap between the requirement and
the availability. Protein is usually the first limiting
nutrient for cattle fed low-quality forages. Most of
the farmers in India feed regionally available protein
meals to the dairy animals, along with other
ingredients. A significant part of these protein meals
is broken down to ammonia in first stomach of
ruminants; therefore, net availability of amino acids
per unit of feed for growth and milk production is
low. However, if these meals are subjected to
suitable chemical treatment – termed as “bypass
protein technology”, then their efficiency of
utilization can be significantly improved. When
chemically treated protein meals replace untreated
one, then due to less degradability of the protein,
excessive loss of both nitrogen and energy could
be avoided, resulting in an increased energy and
nitrogen balance and causing increase in milk yield
and different milk constituents. In a typical diet,
approximately 40% of the protein eaten must be
true protein that escapes degradation, whereas
60% of the protein value can be a mixture of protein
and non-protein nitrogen that is degraded and
incorporated into the rumen microbes (Tarique et
al., 2010). Usually, protein meals are degraded in
the rumen to the extent of 65-70 per cent, leading
to wastage of nitrogen by its excretion through dung
and urine. These protein meals are treated suitably,
so as to reduce their degradability in the rumen from
60-70% to 25-30%, in a specially designed airtight
plant. Cost of treatment of protein meals is less
than a rupee per kg and on feeding one kg treated
meal in comparison to untreated; there is increase
in milk production by more than a liter. Bypass
protein technology is being provided to the dairy
cooperatives and private agencies (Gulati et al.,
2001).
Desirable characteristics for bypass protein
supplements

High level of crude protein

Optimal essential amino acid
profiles

About 70-80% of the protein to be in
9
 a rumen undegradable form

Approximately about 80% of the
rumen undegradable protein to be
digestable in the small intestine
Advantages of bypass protein feed technology
• Higher availability of amino acids per unit of
feed
• Better utilization of those protein meals
having higher rumen protein degradability
• Judicious utilization of protein meals,
available in limited quantity
• Improves growth and milk production
• Improves protein percent in milk, hence,
improves SNF content of milk
•Improves fat percent in milk
• Better economic returns, for same input
cost
• Positively influenced wool growth and quality
• Useful for low and high yielding animals, very
relevant to Indian conditions of feeding and
management
An additional output has been the development of
a slow-release NH3 source, which when used in
combination with by-pass protein feed supplements
lifts milk production a further 5-10 %.
Methods tried for enhancing bypass protein
value of protein meals:
• Formaldehyde Treatment
• Alkali Treatment (NaOH): 1 %, 2 % and
3% supplementation may increase in
rumen bypass ability by 4-5 %.
• Alkali Treatment (NH4OH): 0.3%, 0.5%
and 1.0% supplementation may increase in
rumen by pass ability by 7-8 %.
• Heat Treatment: It is the combination of
LIVESTOCK LINE, MARCH 2012
time and heat which decreases the solubility
of proteins by creating cross linkages both
within or among peptide chains and to
carbohydrates. It is done by two methods-
Jet sploding method and Extrusion method
.In jet sploding method, high temperature
treatment of protein is done at 315oC for
short time. In extrusion method, heat
treatment is done along with steam. But it
has disadvantage that, it causes mallard
reaction and produces melanoidins.
• Oesophageal groove closure:
Oesophageal groove is functional in young
animals and nonfunctional in adult
ruminants. Certain chemicals activate
oesophageal groove in adult animals like
salts of Copper, Silver, Zinc, Sodium etc.
• Tannic acid Treatment: Tannins are
polyphenolic compounds. They have greater
affinity towards proteins. It has been found
that 4% tannin content in diet has increased
the protein and amino acid flow to lower GIT
and are absorbed in lower GIT and improved
the nitrogen retention in animals, thus
concluding 4% tannin can be used as
protein protectant. Condensed tannin
protein complex is insoluble even under
acidic conditions.
• Use of analogs and derivatives of
methionine: Amino acid derivatives are free
amino acids to which a chemical blocking
group has been attached to ””-amino group
or acyl group e.g. Isopropyl DL-methionine,
t-butyl DL- methionine, N-stearoyl DL-
methionine, Capryl-caproylic DL-
methionine. Amino acid analogs are
produced by substitution of ””-amino group
of amino acid with hydroxyl group. Most
commonly studied analog is methionine
hydroxyl analog of 2-hydroxyl-4-
methyliobutanoic acid (HMD)
• Post ruminal Infusion: Proteins and amino
acids are directly infused into abomasum
or duodenum instead of being a part of diet.
Among these, formaldehyde treatment is most
commonly used and economically feasible.
10
 Advantages of formaldehyde treatment in the
production of bypass protein:
Protein sources differ in their rumen degradability.
Some protein meals contain naturally available
rumen bypass protein (30 to 50 % of total CP) viz.
cottonseed meal, toasted soybean, toasted
groundnut meal, maize gluten etc., which can be
used in bypass protein feeds. The cost of these
ingredients is high, whereas, rapeseed meal,
sunflower meal, guar meal etc. are available at
cheaper rate but rumen protein by-pass content in
these meals is low. Such protein meals having high
rumen degradability can be subjected to heat or
chemical treatment for increasing the level of rumen
by-pass occurring. These by-pass protein meals
can enhance the post ruminal supply of critical
amino acids (Prasad and Reddy, 1998). Protein
meals treated with formaldehyde in sealed
chambers, where these undergo formation of
complexes resist degradation in the rumen (Ashes
et al., 1995). The process occurs under
occupational health and safety procedures (Owens
et al. 1990). This attributes to HCHO-binding to the
proteins by formation of methylene bridges
(Fraenkel-Conrat and Olcott, 1948), which makes
them resistant to microbial attack (Walker, 1964).
Different protein meals could be tested for degree
of protection using in vitro procedure to measure
the degree of protection (Ashes et al. 1995, Gulati
et al., 2002; Garg et al.,2004). Treating protein
meals with formaldehyde has the following
advantages:
• Desired level of protein protection can be
achieved.
• Under and over protection of proteins can
be eliminated.
• The bio-availability of the essential amino
acids can be maximized.
• It does not increase the proportion of ADN
and NDN contents.
• Less expensive than heating.
• Helps to control salmonella and reduce
mould growth in feedstuffs.
LIVESTOCK LINE, MARCH 2012
Protein meals treated with formaldehyde to produce
bypass protein should have the following
characteristics: proportion of UDP 70-80%, bio-
available lysine 80-85%, unchanged levels of acid
detergent insoluble nitrogen (ADIN) 2.4-3.0% and
neutral detergent insoluble nitrogen (NDIN) 4.0-
5.0%. These bypass protein supplements could be
included in the diets of lactating cows and buffaloes
for improving milk yield and composition.
Optimization of treatment for protein meals
To avoid over or under protection, protein meals
need to be given optimum chemical treatment, so
that their digestion in the intestine can be
maximized. Maximum protection of protein meals
was obtained at 9-10 days of incubation in airtight
conditions. Lysine and methionine are reported to
be the most limiting amino acids for milk production
(Schwab, 1995; Xu et al., 1998).On protection,
availability of limiting amino acids increased
significantly.
Operational health and safety aspects
Formaldehyde is widely used in industry and occurs
naturally as a constituent of many foods including
dairy and meat products, coffee, fruits, smoked fish
e.g. 0.2 ìg/g in meat; 0.1 ìg/g in milk; 10 ìg/g in
cheese; 180 ìg/g in fish (Owens et al, 1990).
Formaldehyde is converted to formic acid by the
action of the formaldehyde dehydrogenase enzyme;
formic acid is metabolized to carbon dioxide and
water, or incorporated into the one carbon pool or
excreted in the urine as a sodium salt (Owens et
al, 1990). Hence, mammalian systems have the
biological pathways to effectively metabolize
ingested formaldehyde and there is no evidence to
suggest that formaldehyde is a carcinogen when
consumed orally (FDA, 1998). The formaldehyde
present in treated feedstuffs is metabolized by
ruminants and does not significantly change the
naturally occurring levels of formaldehyde in meat
and milk (Atwal and Mahadevan, 1997).
Formaldehyde is approved for use as a feed
additive to protect proteins from ruminal degradation,
to preserve silages, to maintain animal feeds or feed
ingredients free of salmonella, to control fungi and
to improve the handling characteristics of oilseeds
and meals, and animal fat pre-mixes (FDA, 2004).
For treatment of protein meals, level of
11
 formaldehyde used is not more than 0.8 per cent. After two days of incubation, formaldehyde level in
protein meal is detected below 2 ppm. So, handling of treated protein meals is not a serious problem
from animal and consumer health hazard point of view.

Table 1: Level of essential amino acids available for absorption in unprotected and protected protein meals:

Source: Gulati et aal., 2002

LIVESTOCK LINE, MARCH 2012 12

 Reference
Ashes, J.R., Gulati, S.K., and Scott, T.W. (1995).
The role of rumen protected proteins and energy
sources in the diet of ruminants. In: Animal Science
Research and Development (Ed. Ivan, M). Centre
for Food and Animal Research Agriculture and Agri-
Foods, Canada, pp.177.
Atwal, A. S. and Mahadevan, S. (1997).
Formaldehyde in milk not affected by feeding
soybean meal coated with chemically treated zein.
Canadian Journal of Animal Science, 74: 715-716.
FDA. (2004).‘Food Additives Permitted in Feed and
Drinking Water of Animals: Formaldehyde.’ Food
and Drug Administration, Department of Health and
Human Services, Washington, DC. 21CFR Part
573 (Docket No. 1998F-0552).
Fraenkel-Conrat, H. and Olcott, H.S. (1948).
Reaction of formaldehyde with proteins. Cross
linking amino groups with Phenol, Imidazole or
Indole group. Journal of Biological Chemistry,
174:827.
Garg, M. R., Sherasia, P. L, Bhanderi, B. M., Gulati,
S. K. and Scott, T. W. (2002). Effect of feeding
rumen protected nutrients on milk production in
crossbred cows. Indian Journal of Animal Nutrition,
19 (3):191-198.
Garg, M. R., Sherasia, P. L., Bhanderi, B. M., Gulati,
S. K. and Scott, T. W. (2004). Effect of feeding
protected protein on milk production and
composition of lactating cows. Indian Veterinary
Journal, 81(1): 48-50.
Garg, M.R. (1998). Role of bypass protein in feeding
ruminants on crop residue based diets. Asian-
Australasian Journal of Animal Sciences, 11: 107-
116.
Gulati. S.K., Ryde. I., Kaur. R., Scott. T.W., Garg.
M.R., Serasia P.L. and Singh D.K. (2001). Role of
protected nutrients in sustainable milk production.,
In Proc. X Animal nutrition conference. Karnal, India.
U
LIVESTOCK LINE, MARCH 2012
Gulati, S.K., Scott, T.W., Garg, M.R. and Singh, D.K.
(2002). An overview of rumen protected or by-pass
proteins and their potential to increase milk
production in India. Indian Dairyman, 54: 31-35.
Owens, B.A., Dudney, C.S., Tan E.L. and Easterly,
C.E. (1990). Formaldehyde in drinking water:
Comparative hazard evaluation and approval to
regulation. Regulatory Toxicology and
Pharmacology, 11: 220-236.
Patton, R.A. 2009. The Strategic Use of Ruminally
Protected Amino Acids in Dairy Nutrition. d.
ifas.ufl.edu
Prasad, P.E. and Reddy, R.R. (1998). Effect of
formaldehyde treated groundnut cake on in vitro and
in sacco protein degradability. Indian Journal of
Animal Nutrition, 15: 52-54.
Schwab, C.G. (1995). Rumen protected amino
acids – their role in nutrition of high producing dairy
cows. In: Animal Science Research and
Development: Moving towards New Century. (Ed.
Ivan, M.) Ottawa, Canada.
Taquire N.A., Shahzad M.A., Nisa M., Sarwar M. and
Fayyaz M. 2010. Influence of bypass protein on
Buffalo productivity. Proceedings 9th world buffalo
congress. New Delhi, India.
Walker, J.F. (1964). Formaldehyde. 3rd Ed. Reinhold
Publication, New York (Fide McDonald, I.W. 1968).
Nutritional Aspects of Protein Metabolism in
Ruminants. Australian Veterinary Journal, 44:145.
Walli, T.K. (2005). Bypass protein technology and
the impact of feeding bypass protein to dairy animals
in tropics: A review. Indian J. Anim. Sci. 75 : 135-
142.
Xu, S., Harrison, J.M., Chalupa, W., Sniffen, C.,
Julien, W., Sato, H., Fuvieda, T., Watanabe, K.,
Veda, T. and Suzuki, H. (1998). The effect of ruminal
bypass lysine and methionine on milk yield and
composition of lactating cows. Journal of Dairy
Science, 81: 1062-1077.
13
 A Few Nutritional Updates for
Feeding Dairy Animals
Dr Trishna B. Kayastha & Dr Sanjeeb Dutta
Department of Animal Nutrition & Livestock Production and ManagementApollo College of
Veterinary Medicine, Jaipur-31 Mobile phone no 09602564270.
U
India is the highest milk production country in the
world and by the end of 2022, India aims at
producing 172 MMT milk at an annual growth rate
of 4 per cent, but its cattle feed industry has not
kept pace ahead. There is a need to change the
conventional system of preparing cattle feed so as
to meet the current challenge of high yielding cross
bred cows and improved buffaloes. The bio-mass
resources is very limited and there is shortage of
feed and fodder resources in the country. So the
available feed resources would need to be utilized
judiciously and with value addition. Farmers
therefore need to be encouraged to adopt improved
and balanced feeding practices so that they could
improve yields with available feed resources in a
cost effective manner. In this way milk out put could
be doubled.
Feeding plays vital role in exploiting the genetic
potential of dairy animals. Feeding is also
considered critical in the overall success of dairy
development program, as feeding alone consists
more than 70% of the total cost of milk production.
Indian cattle industry is an integral part of Indian
agriculture and contributes to the well being of its
people. Over 80% of these livestock unit is
ruminants and can be largely described as grass
eater while the rest is monogastric grain eaters.
Presently India is bestowed with a huge livestock
population comprising 222 million cattle, 98 million
buffaloes, 124 million goats, 61 million sheep and
489 million poultry. Animal Husbandry, Dairy and
Fisheries generate supplementary incomes and
gainful employment for rural house holds,
particularly among landless, marginal or small
farmers, as well as women in one hand and the
products obtain from these sectors are also a
source of valuable nutrients to millions of people in
India. Dairying only provides nearly 2/3 of the total
livestock’s contribution to GDP with an encouraging
growth rate of 5 percent. Agriculture and allied
sectors account for about 24% of GDP. Of this,
animal husbandry and dairy accounts for about
LIVESTOCK LINE, MARCH 2012
25%. More than 70 million rural families are
engaged in milk production in India. Landless, small
and marginal farmers with limited resources
account for 65% of the total milk production in the
country. Dairy cattle production is mostly based on
crop residues such as straws, stovers and agro-
industrial byproducts. Over the years there has
been a perceptible change in total livestock
population. The milch buffaloes and crossbred
cattle population has been increasing gradually,
while the male population of cattle and buffaloes
have been decreasing due to mechanization in
agriculture and shortage of feed resources for
feeding unproductive animals. Feed resources can
be broadly categorized into dry fodder (crop
residues), green fodder and concentrates. Crop
residues include mostly wheat, paddy, sorghum and
millet straws, kabdies etc; green fodder include
cultivated legumes and non-legumes, pastures,
sugarcane tops etc and concentrates include
grains, oil cakes/meals, brans, chunnies, and agro-
industrial by-products. There always exist a huge
shortage of concentrate and green fodders that has
been calculated using appropriate grain to straw
ratios for crop residues, extraction rates for
concentrates and average green bio mass
production potential for different categories of land.
To minimize the gap between requirement and
availability of feed resources, some of the
technologies that could be used in utilizing feed
resources judiciously with value addition are briefly
describe below.
1. Implementation of Ration balancing program.
2. Feeding of Compound Cattle Feed.
3. Supplying protein source of feed in the form of
by pass protein.
4. Supplementation of area specific mineral mixture.
5. Feeding of Urea Molasses Mineral Block Lick.
6. Enrichment and Densification of Crop Residues.
7. Enhancement of Green Fodder Production.
Implementation of Ration balancing program
14
 In Indian dairy animal ration only 10% of total feed
ingredients are provided in the form of compounded
cattle feed. The rest of the feed ingredients in the
ration comprise with locally available or home grown
feed ingredients as such or some farmers used
only brans, grains or cakes to feed their animal.
These ingredients are rich in only one or two
nutrients. For eg brans are rich source of
Phosphorus and low in Calcium. But animals
respond better in terms of growth and milk
production when they receive all the major as well
as minor nutrients in right proportion “so called
balance ration” as per their body’s requirement that
can only be obtain by preparing ration with
ingredients like energy rich eg;- cereal grains;
medium energy & medium protein eg: choker
churies and brans; protein rich eg: oil cakes and
other agro-industrial byproduct that are locally
available with more or less similar or lower input
cost. These are mixed in right proportion and
fortified with small quantity of mineral and vitamin.
So, it is very much essential to implement the ration
balancing program at the farmers door step so that
such type of balance feeding can fully exploit the
genetic potentiality of the dairy animal. NDDB has
developed computer software for ration balancing
program. In this training program farmers are
advised to feed their animals a balance ration which
is computed by computer based least cost
computation and which takes into account the
animals nutrient requirement according to different
physiological conditions as well as the nutrient
available to the animal from the prevailing feeding
practices.
Introducing Compounded Cattle feed for dairy
animals
The compounded feed is a blend of all feed stuffs
ie fodder, cereal grains, oil seed cakes, brans,
churies, agro-industrial byproducts, mineral and
vitamins that are thoroughly mixed in suitable
proportion which provide adequate nutrients to meet
the needs of dairy animals. Each bite consumed
provides the required level of nutrients needed by
the animals. With the feeding of compounded feed
along with basal diet, nutrient requirement of animal
can be met more efficiently and economically, but
this needs proper grouping of animal according to
physiological stage and production level. For
example high energy ingredients can be liberally
fed to high producers kept in the separate group
LIVESTOCK LINE, MARCH 2012
without overfeeding to the late-lactation. It provides
greater flexibility in feeding exact amounts of
nutrients as per level of milk production. Thus
compounded cattle feed system of feeding can
save labour and reduce overall feeding cost. The
Bureau of Indian Standards (BIS) have been
prescribe two categories of compounded cattle feed
Type I (containing min 22% CP, min 3% EE, max7%
CF & max 3% AIA) and Type II (containing min 20%
CP, min 2.5% EE, max 12% CF & max 4% AIA)
which are suitable to feed the cattle or buffalo
yielding more than 10 liters of milk per day. But a
majority of dairy farmers in India possess two or
three cows/buffaloes yielding about 2 to 4 liters of
milk per day. The nutritional parameter prescribed
for these two type of cattle feed do not allow the
incorporation of abundantly available agro-industrial
by product so these are cost effective. In order to
encourage the majority of the farmers to use
compounded cattle feed CLFMA(Compounded
Livestock Feed Manufacturer Association ) felt
necessity to introduce additional categories to feed
animals of different production capacities based on
milk yield and body weight as for High yielder Type
I (containing min 20% CP, min 2.5% EE, max 7%
CF & max AIA 4%), for medium yielder Type II
(containing min 18% CP, min 2.5% EE, max 12%
CF & max AIA 4.5%) and for low yielder Type III
(containing min 16% CP, min 2% EE, max 14% CF
& max AIA 5%).
Practice of Feeding Bypass protein
supplements
The availability of protein source feed for animal in
India is very limited. Usually about 70%
proteinacious feed is degraded in the rumen to
ammonia by the ruminal microbes and significant
portion of it is excreted out in the form of urea
through urine. However, if such type of proteinacious
feed is subjected to suitable chemical treatment
termed as “bypass protein technology”, their
efficiency of utilization can be significantly improved.
NDDB has standardized and commercialized
bypass protein technology, using locally available
proteinacious feed such as groundnut cake,
mustard oil cake, sunflower cake, guar meal etc.
These proteinacious feeds are treated suitably, so
as to reduce their degradability in the rumen from
60-70% to 25-30% in a specially designed airtight
plant. Cost of bypass protein supplement is less
than a rupee per kg and on feeding one kg treated
bypass protein supplement; there is increase in
15
 milk production by more than a liter in comparison to untreated proteinous feed, which is well established
after several feeding trial in dairy animals. The specifications of bypass protein feed produced in NDDB
on dry matter basis is min 30% CP, min 3.5% EE, max 8% CF, max 2.5% AIA, min 20% UDP & max 9%
RDP. The overall benefits of feeding bypass protein to the dairy animals are summarized as follows.
1) Higher availability of amino acids per units of feed.
2) Better utilization of proteinacious feed having higher rumen protein degradability.
3) Judicious utilization of protein source available in limited quantity.
4) Improves growth and milk production (0.8-1.2lit/day)
5) The percentage of protein in the milk increases (0.1-0.3%), hence improves SNF content of milk.
6) The percentage of fat in the milk increases (0.2-0.8%).
7) Better economic returns from same input cost.
8) Useful for low and high yielding animals, relevant to Indian condition of feeding and management.

Supplementation of Area Specific Mineral Mixture

In India, crop residues are used as staple feed for dairy animals which are very poor in essential minerals
as it contains several anti-nutritional factors like silicate, oxalate, phytic acid etc that further inhibit the
utilization of several minerals. As animals can not synthesize minerals inside their body, supplementation
of mineral mixture with their ration is outmost important. Minerals both macro (Ca, P, Na, K, Cl, S, Mg) as
well as micro (Zn, Fe, Cu, Co, I, Se, Mn etc) are equally play important role in growth, production,
reproduction and many metabolic activities of the body. In India BIS has recommended two types of
mineral mixture Type I (Containing salt) and Type II (without salt) for supplementing cattle feed, the
characteristic of which are as follows:-

Implementation of Urea Molasses Mineral Block lick (UMMB)

UMMB are the lick blocks containing urea, molasses, vitamins, minerals and other multinutrients. The
feeding of the block is a convenient and inexpensive method of providing all the nutrients required by
both the rumen microbes and the animal which may be deficient in the diet. A standard UMMB consists
of molasses (30-50%), urea (5-10%), a cereal bran such as rice, wheat or maize bran (15-25%), an oil
cake (10-12%), salt (5-7%), lime or calcium carbonate (5-10%), bone meal (5-7%) and trace mineral
mixture (1-2%). Urea is only source of nitrogen (46%) which readily hydrolyzed in the rumen producing
ammonia. So readily available source of energy such as molasses (cheap source must be given in urea

LIVESTOCK LINE, MARCH 2012 16

 supplemented feed so that the rate of hydrolysis of
ammonia from urea and release of energy from
the molasses remain constant. As a result the
rumen microbes can easily form the microbial
protein for the host animal without causing
ammonia toxicity. Such multinutrient block not only
provides overall nutrient requirement of ruminant
animal but also these are more convenient for
packaging, storing and transportation.
However the consistencies of the molasses play
an important role in the successful manufacture of
UMMB which depend upon the quantity of sugar in
the molasses. This sugar quantity, expressed as a
percentage of total weight in the molasses is called
the BRIX value. To ensure good hardening the BRIX
value should be 80 or more that can be checked
with a small pocket refractometer.
Enrichment and Densification of Crop
Residues
The availability of crop residue all over India
throughout the year is not uniform, with some areas
having a surplus and others facing a perennial
shortage of dry fodder. Regional imbalances and
shortages of crop residues lead to Sub-optimal
livestock productivity due to imbalance feeding and
significant cost on account of transportation. In
India, major part of basal ration of ruminants
constitute crop residue. So, there is a need to
manage feed and fodder resources efficiently with
value addition. In areas where crop residues are
deficit are sold @ Rs 4/kg, on the other hand they
are often burnt in surplus areas. If crop residues
are enriched and densified in the form of blocks,
pellets, briquettes etc, these can be transported at
lower cost from the surplus to deficit regions.
Some examples of Straw-based pellet and Straw-
based block produced by NDDB are respectively
as follows
Wheat straw-40%, Deoiled rice bran-37%, Mineral
mixture-1%, Common salt-1%, Rice polish fine-5%,
Urea-1%, Molasses-15%. & Straw-64%, Deoiled
rice bran- 20%, Urea-1% and Molasses-15%.
Such multinutrient UMMB provide approximately 8-
10% CP and 55% TDN.
Enhancement of Green Fodder Production
The fodder based systems of feeding help to lower
feed costs, but the scope for such system is limited
LIVESTOCK LINE, MARCH 2012
in India because of the need to give priority to food
crop. The average cultivated area under fodder crop
is estimated as 4.4% ie around 9.38 million hector.
In areas with better irrigation facilities, intensive
fodder production is practiced in the Northern
Region particularly Punjab and Haryana where 10%
of the irrigated land is allocated for fodder cultivation
(mainly Lucerne, berseem, maize, oat, sorghum
etc). As limited land is available for green fodder
production, fodder yield per hector can be improved
by (a) Supplying farmers certified fodder seeds, to
cover at least 10% of the total area for fodder
production, (b) Efforts should be made to develop
wasteland through watershed management for
green fodder production, so that the gap between
the requirement and the availability could be
minimized. NDDB has designed and implemented
fodder seed multiplication and distribution project
through dairy co-operatives. NDDB is assessing
milk co-operatives in the procurement of breeder
seeds, its multiplication by the farmers and
establishing fodder seed processing plants.
Conclusion
Keeping in view the wide gape of the availability of
feeds and fodder for the livestock as per their
requirements, nutrition research should emphasize
the development of feeding systems based on
existing feed resources under farm conditions.
There should be a feed security system for animals
needs to be developed to meet the requirements
of livestock in famine and for draught or flood prone
areas. Further introduction of cultivated fodders
having high yield and identification of non-
conventional feeds for livestock and developing
processes for improving their nutritive values needs
to be undertaken on large scale.
References
Dr D.D.Sharma. (2002). Scope for using complete
feed blocks for increasing dairy production in India.
All India Dairy Husbandry Officers’ Workshop (26-
27) th November, 55-61.
David J. Schingoethe. (2010). Feeding dairy cows.
Livestock Feeds and Feeding. 306-322.
Dr D.V. Reddy, Nutritional Requirement of Indian
Cattle and Buffalo. Applied Nutrition. Pp 53-104.
Richard O., Kellems and D.C. Church. (2010)
Supplemental Protein Sources. Livestock Feeds
and Feeding. 84-113.

17
 COMMON DISEASES OF LIVESTOCK CAUSED BY
BACTERIA AND THEIR DIAGNOSIS
*
Phaniraja.K.L., Prasanna.S.B and Ravikumar .C
*
Dept. of Veterinary Microbiology, Veterinary College, KVAFSU, HASSAN- 573 118
U
TUBERCULOSIS
Tuberculosis (TB) remains as a world wide public
health concern despite its agent was established 100
yrs ago and efficient drugs and vaccines are
available since then. Even today the world records
an occurrence of 7.25 million new cases and 3 million
deaths every year due to TB. It is a chronic infectious
disease of animals and also Humans caused by an
acid fast bacilli Mycobacterium bovis, a facultative
intracellular pathogen with an ability of resisting
intracellular killing by phagocytes. Bovine TB
organism is closely related to Mycobacterium
tuberculosis in nucleotide sequence, with a high
degree of antigenic relatedness. . and slaughter” which itself is creating a big window
Factors contributing spread of bovine TB:
Bovine TB gains its utmost importance because of
its relatedness to Mycobacterium tuberculosis and
its ability to cause similar disease in human beings
causing a significant public health crisis. TB caused
by M.bovis is clinically indistinguishable from that of
M.tuberculosis. Information on human disease by
M.bovis in developed and developing countries is
scarce. However, the abstract of several Zoonotic
TB studies carried out around the world indicate the
proportion of human cases due to M.bovis accounted
for 3.1% of all forms of TB; 2.1% of pulmonary forms
and 9.4% of non pulmonary forms.
Since the disease is zoonotic, risk factors
contributing zoonosis log on to both animals and
human population..
A. Animal risk factors:
1. Constant source: The distribution of M.bovis in
domestic and wild animal population represents a
huge pool of infectious agent, even in countries which
had totally eradicated the disease; it is again on a
rise because of wild reservoirs, where in U.K is a
great example with badges being the source of
reinfection after almost eradication.
2. Milk production and animal husbandry:
Introduction of more productive cross breed /exotic
animals leading to white revolution has made India
today the highest milk producer in the world. It is a
well known fact that indigenous animals are resistant
to tuberculosis. Bovine TB is most common in
organized dairy herds especially with more numbers
of crossbreeds/exotic animals (in urban areas).The
LIVESTOCK LINE, MARCH 2012
intensive husbandry activities with poor management
and overcrowding are playing a major role in the
spread of the disease.
3. Inadequate control programmes: The basic
strategies required for control and elimination of
Bovine TB are well established however due to
financial constraints, scarcity of trained
professionals, lack of political will, as well as the
underestimation of importance of TB in both animal
and public health by successive Governments.,
control measures are applied inadequately or not
at all put into practice . Presently we have got a “test
and isolate” policy against the global policy of “test
for disease spread.
B. Risk factors from Human population:
1. Physical contact : In India cattle are very much
an integral part of human social life, hence there is
close contact between man and potentially infected
animals, with a very high risk human infection.
2. Food hygiene practices : In principle, milk from
affected animals has been regarded as principle
source of M.bovis to humans. In areas where bovine
TB is endemic, milk borne infection is the main cause
of cervical lymph adenopathy and other forms of non
pulmonary TB.
In India, of late, a huge competition has been
created by large scale state run, marketing
enterprises and the informal sector. The informal
sectors can ignore standards of hygiene and quality,
which could be a direct source of infection to the
consumers.
3. HIV /AIDS: It has been well established that TB is
the most frequent opportunistic disease along with
HIV infection. Persons with both infections have an
annual risk of progression to active TB of 5% to15%
depending on their level of immunosupression.
LABORATORY DIAGNOSIS:
A. Single Intra dermal PPD testing in cattle/
Tuberculin Test: involves injection of 0.1.ml
of PPD at the neck or caudal fold region of
the animal. If erythema and induration follow,
the test is considered as positive.
B. Demonstration of organisms by acid fast
staining.
18
 C. Postmortem and histopathology.
D. Cultivation of the organisms on primary
isolation medium.
E. Molecular and Nucleic acid recognition
methods: RFLP, IS- printing and PCR has
been widely evaluated for the detection of
M. tuberculosis complex in clinical samples.
F. Serological Diagnosis by ELISA, IFN analysis.
Control / Eradication : Bovine TB does not often
justify the emergency measures required for other
diseases (e.g., Rinderpest, Leptospirosis, and foot
and mouth disease). The full economic implications
of zoonotic TB are, however, overlooked in India
where the overall impact of the disease on human
health and animal production needs to be assessed.
The present concept of TB control is by testing and
isolating the positive animals in Goshalas. This
concept, instead of reducing the disease incidence
it facilitates increased disease spread. It’s high time
to strongly incorporate the policy of “test and
slaughter”. In the name of ethics and socio religious
reasons, that cattle should not be slaughtered, we
are making the infected animal to suffer from the
disease for a longer period which is also a cruelty in
real sense. In an era, where Governments across
the globe are permitting the human euthanasia in
totally incurable conditions to avoid further suffering,
we have to think seriously in these lines in order to
eradicate the disease from our country.
Following may be the practical and field oriented
concept of Tuberculosis eradication.
1. Test by single intra dermal tuberculin test (or any
other better tests, if feasible)
2. Immediate culling of the positive animals from
the herd by humane slaughter.
3. Careful management of above listed risk factors.
4. Development of an Anti TB vaccine – this is most
relevant today because it was M.bovis strain that is
used as BCG for human TB control with satisfactory
results across the world. Research and development
is the need of the hour in this direction to develop a
better, new generation vaccine for animal use. Even
if the cost of this is in terms of few crores it is much
smaller when compared to total annual economic
loss due to Bovine tuberculosis apart from immense
zoonotic significance the bacteria posses which can
never be assessed financially.
JOHNE’S DISEASE / PARATUBERCULOSIS
Paratuberculosis, popularly known as Johne’s
disease, is an infectious disease caused by
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Mycobacterium avium sub species Para tuberculosis.
It is primarily a disease of domestic and wild
ruminants, the disease also been reported in horse,
pigs, deer and recently in rabbits and fox. The
disease causes heavy economic losses to the dairy
industry in terms of reducing milk loss and treatment.
The disease is characterized by dehydration,
emaciation, chronic diarrhea and thickening of the
intestine (corrugation). Under natural conditions, the
disease in cattle spreads by ingestion of
M.paratuberculosis from the contaminated
environment. The disease persists after the
introduction of infected animals. Infection can be
spread vertically to the fetus and semen can be
infected with the organism. The primary source of
infection in claves is milk from infected cows or milk
that is contaminated with the feces of diseased cattle.
Transmission and pathogenesis: Feces
containing the organisms are primary source of
infection which is acquired by ingestion of
contaminated feed and water. It has very long
incubation period (15-18 months). The organisms
have also been isolated from genitalia and semen
of infected bulls. Following infection, organism
penetrates the intestinal mucosa and sets up
residence within macrophages. The organism
multiplies intracellular without killing host cells and
are resistant to intracellular digestion. They grow
inside macrophages and distributed throughout the
body. The primary site for bacterial multiplication is
terminal ileum and the large intestine leading to
decreased absorptive surface, chronic diarrhea and
mal absorption.
Early lesions occur in the walls of the small intestine
and the draining mesenteric lymph nodes, and
infection is confined to these sites at this stage. As
the disease progresses, gross lesions occur in the
ileum, jejunum, terminal small intestine, caecum and
coon and in the mesenteric lymph nodes.
Mycobacterium paratuberculosis is present in the
lesions and terminally, throughout the body. The
intestinal lesions are responsible for a protein leak
and a protein mal absorption syndrome, which lead
to muscular wasting. Clinical sings usually first
appear in young adulthood, but the disease can
occur in animals at any age over 12 years.
The infection progress and the animal still does not
show any clinical sings. Nevertheless, the organisms
are being excreted in very high numbers, probably
enough to infect other animals in contact. Infection
is detectable by fecal culture techniques but not
often blood tests. In later stage the animal show early
signs of disease and most diagnostic tests can detect
the infection.
19
 LABORATORY DIAGNOSIS:
A. The cultural examination of feces and direct
microscopic examination of acid fast stain reveals
the presence of organisms like clumps (three or
more organisms) of small (0.5-1.5ìm), strongly
acid fast bacilli are found. The presence of single
acid fast bacilli in the absence of clumps does
not indicate definitive diagnosis. The
disadvantage of this test is that only about one
–third of cases can be confirmed of microscopic
examination of a single faecal sample.
Mycobacterium paratuberculosis infection mainly
involves the lower small intestine and adjacent
caecum. Mycobacterium paratuberculosis
organisms are vastly outnumbered by other
bacteria in faecal and intestine tissue specimens.
The commonly using medias are Herrold’s egg
yolk medium with mycobactin and modified
Dubo’s medium.
B. Using single intradermal PPD Johnin has been
used for many years for screening of animals in
herds. After 72 hrs of infection, it is examined
for the presence in thickness 4mm or above for
positive.
C. ELISA is reported to be most sensitive and
specific test for serum antibodies to M.
paratuberculosis. Its sensitivity is comparable
with that of the CF test in clinical cases, but is
greater than that of the CF test in sub clinically
infected carriers.
D. CFT: the CF test has been the standard test used
for cattle for many years. The CF test works well
on clinically suspect animals, but does not have
sufficient specificity to enable its use in the
general population for control purposes.
Control: Following may be the practical and field
oriented concept of Johne’s disease eradication.
1. Test by single intra dermal Johnin test (or any
other better tests, if feasible) 2. Immediately
cull the positive animals from the herd by humane
slaughter.
BRUCELLOSIS
Brucellosis is an economically important reproductive
disease of livestock including cattle, buffalo, sheep
, goats and pigs. The disease induces infertility,
delayed heat, interrupted lactation, loss of calves,
wool, meat and milk production and is of zoonotic
importance in developing countries, including India.
In India, the abortions in livestock due to brucellosis
was first reported as early as in 1918. Subsequently,
the serological and cultural evidence of infection in
livestock and human beings have been reported from
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various states in the country, including Karnataka.
There is concern that the disease may further flare
up due to intensive dairy development programmes.
In this context, an indepth understanding of the
epidemiology of brucellosis is required in view of it’s
trans host transmission and institution of practical
strategies to control the disease. This paper
highlights the aspects of epidemiology and control
of bovine brucellosis in Karnataka.
Serological survey in bovines:
Seroprevalence studies form the backbone of
epidemiological investigations and have been used
to identify brucellosis-infected herds. Generally, the
cases of reproductive failure and abortion are
screened for brucellosis by RBPT and SAT. During
late 1990s, serum and milk based ELISA kits
developed at Project Directorate on Animal Disease
Monitoring and Surveillance (PD_ADMAS),
Bangalore found wide acceptance. Milk based ELISA
was preferred for screening pooled milk samples at
village based milk co-operative societies. The details
of brucellosis survey is dealt elsewhere.
Brucellosis control:
The Govt. of India has a centrally sponsored scheme
on brucellosis control in most of the states including
Karnataka, in the name of Systematic Control of
Livestock Diseases of National Importance. The
functioning of this programme are mostly restricted
to serological survey and suggesting the livestock
owners to send the sero-positive animals to
Goshalas. The cost involvement in maintenance of
such infected and unproductive animals is high and
is economically not feasible. However, due to lack
compensation, such brucellosis positive animals are
sold due to distress instead of being sent to
Goshalas. On the other hand, the ongoing individual
animal screening and declaration of diseased
animals could be partially responsible for spread of
disease. Thus this programme appears to be
counter-productive and has hardly contributed much
to any approach towards disease control.
Furthermore, cow slaughter is banned on religious
basis. In addition, the commercialization of dairy
sector provides for frequent movement of animals.
However, it is practically difficult to restrict the
movement of such animals not only within the state
but also at the inter state level.
Recently, Govt. of India has directed that all the bulls
used in the production of semen for artificial
insemination purpose should be regularly tested and
strictly brucellosis free bulls to be used for semen
collection. It has also identified Regional Disease
20
 Diagnostic Laboratory for this purpose (Southern
Regional Disease Diagnostic Laboratory, SRDDL in
Karnataka). With this directive, practically all the bull
farms in the Govt. and Co-operative sectors are
subjected to regular testing and affected bulls are
being culled. Due to this initiation, breeding bulls in
these semen stations may no longer serve as source
of infection to inseminated cows.
In Karnataka, vaccination against bovine brucellosis
is not generally practiced except in some infected,
organized, private and military dairy farms. In
general, Calf hood vaccination with B. abortus S-19
is practiced in a meager scale. Vaccination of calves,
the elimination of reactors, improved herd
management and zoosanitary measures are
recommended for effective control of brucellosis. An
awareness programme needs to be introduced to
highlight the public health importance of this disease.
Brucellosis should be controlled by vaccination of
bovines together with other measures such as
movement control and testing and isolation of
infected animals. This eventually reduces the
transmission to human beings. While the effective
control measures are still need to be implemented,
veterinarians and other workers may enlighten the
public, villagers, risk groups to prevent brucellosis
by boiling of milk, avoid consumption of
unpasterurised milk. Farmers should know that
vaccination of their animals for brucellosis is
important. Vaccination of livestock is relatively cheap
and will increase the value and productivity of their
animals.
Brucellosis in sheep and goats in the state, which is
mostly responsible for brucellosis among
veterinarians and Inspectors, is also reported by
several researchers including both veterinary and
Medical professionals. Generally it is believed that
B. abortus infects bovines and B. melitensis infects
small ruminants. However, isolation of B.melitensis
from Jersey cross bred cows of the dairy farm
belonging to a semi Govt. organization in 1987 was
of major concern. The authors postulated that the
infection from sheep and goats in the same farm
was transmitted to cattle through contaminated
fodder or some other vehicle, as livestock species
such as cattle, buffalo, sheep and goats in the farm
had access for common grazing. Thus, free grazing
and movements with frequent mixing of flocks of
sheep and goats with cattle also contribute to the
high prevalence and wide distribution of brucellosis
in these animals. This situation needs to be viewed
seriously from public health point of view as
B.melitensis infections, than B. abortus, are most
commonly encountered in human patients. Therefore
LIVESTOCK LINE, MARCH 2012
there is an urgent need for the strict implementation
of a control policy not only for cattle but also for
small ruminants. Unfortunately, non-availability of
vaccine locally for controlling brucellosis in sheep
and goats is a major limitation. The situation
demands it’s immediate availability for the local use
in view of controlling trans host transmission of
brucellosis from small to large ruminants and also
reducing the zoonotic impact of the disease.
BLACK QUARTER
(BLACK LEG / INFECTIOUS MYOSITIS)
True Balckleg / Black Quarter is the Clostridial
myositis of skeletal muscles caused by Clostridium
chauvoei (Cl. feseri), a Gram positive, spore-forming
rod-shaped bacterium. Disease is common only in
cattle but occurs in other animals under traumatic
conditions.
Epidemiology: Cattle in age group of 6 months to
2 years that are rapidly growing with high nutritional
status are affected. Occurs in warm wet months,
spring to autumn, high rain fall, excavation of soil
which exposes and activates spores of the causative
agent and the mortality goes upto 100 per cent. It is
a soil-borne infection. Portal of entry is probably
through the alimentary mucosa by contaminated
feed or during erupting teeth. Bacteria may be found
in the spleen, liver and alimentary tract of normal
animals. Contamination of soil and pasture occur
from infected feces or carcasses of affected animals.
Disease develops by invasion of tissues by
organisms during trauma or anoxia. In cattle the
disease occurs without history of trauma but in sheep
it is always a wound infection during shearing,
lambing, fighting, during vaccinations with tissue
damage or fetus of infected ewes.
Pathogenesis: Trauma or unnoticed trauma may
trigger the disease. Toxins(á,â,ã and ä) formed by
the organism produces necrotizing myositis locally
in skeletal muscles and systemic fatal toxaemia. In
cattle and sheep atypical outbreaks of sudden death
occurs in which the lethal lesion is a clostridial cardiac
myositis.
Clinical findings: Lameness, pronounced swelling
of upper limb, myonecrosis of skeletal or cardiac
muscles, high temperature (106o F) and pulse rate
(100-120 / min), severe toxaemia, depression,
ruminal stasis, anorexia, gaseous crepitating
emphysema, discolored, dry and cracked lesions.
Animals may be found dead with high mortality.
Lesions may vary slightly in sheep and horses.
Necropsy findings: Generally myositis, dark
coloured, rancid odour, metallic sheen on the cut
21
 surface of affected muscle are seen. Cattle are found
dead lying on the affected side of the hind limb with
stiffness. Bloating and putrefaction occur quickly.
Blood-stained froth exudes from the nostrils and
anus. Blood clotting occurs rapidly. In sheep lesions
are not so marked as in cattle.
LABORATORY DIAGNOSIS: Smears from affected
tissues are collected for bacteriological examination
as soon as possible. Isolation and identification of
Cl chauvoei and Cl novyi is difficult due to their
fastidious nature and due to clostridial post-mortem
invaders from the gastro intestinal tract.
1. Muscle pieces collected under aseptic
precautions are kept in sterile air-tight
container and sent to laboratory as quickly
as possible for anaerobic culture
2. Four air dried impression smears for FAT and
microscopic examination.
3. Culture from needle biopsy or swabs from
wounds.
Serological test: By fluorescent antibody test (FAT)
for tissue smears
Control: Annual vaccination of all cattle between
3-6 months with 2 vaccinations given 4 weeks apart
and annual booster vaccination done prior to risk
period. In an outbreak all unaffected cattle should
be vaccinated immediately and treated with 10,000
units / kg penicillin, I/M. Segregation, early treatment
are beneficial. Clostridial vaccines have poorer
antigenecity in sheep and goats than in cattle.
Proper disposal and zoo-sanitary measures like
deep burial of dead carcasses and Infected materials
with maintenance of hygienic conditions of the
premises. Inactivated alum-precipitated vaccine
is widely used in the country with satisfactory results.
ANTHRAX
Anthrax is an important zoonotic disease primarily
affecting large domesticated animals and infects man
accidentally through contact with infected animals
and animal products. In natural conditions, both wild
herbivores and domestic animals are highly
susceptible and birds are resistant to the disease.
Bacillus anthracis is the causative organism, is of
world wide distribution. Repeated outbreaks occur
in Asia, Australia, Africa, Southern Europe &
Southern America. It has roughly 1,200 various
strains. It is a gram positive large rod, non motile,
capsulated, non haemolytic bacillus, occurring in
short or long chains.and can take two forms: the
vegetative bacilli and the spore. B. anthracis is more
dependent on sporulation for species survival
LIVESTOCK LINE, MARCH 2012
making it an obligate pathogen. The spores are
resistant to chemical disinfectants and heat.
However, autoclaving at 121° C at 15 lbs pressure
destroys them in 15 minutes. The infection is
transmitted by the spores of the bacillus, which are
shed in large numbers in the terminal stages of
infection. The disease is therefore unusual in that
the infection is spread only from dying or dead host
and that the causative organism may survive for a
long time in the environment.
Pathogenesis and Virulence determinants: The
pathogenicity depends on two properties of the
organism that are not found in saprophytic Bacillus
species. The most easily demonstrated determinant
is the capsule which is unusual in being a
polypeptide, of D-glutamic acid which inhibits
opsonophagocytosis. The other determinants are
the potent exotoxins comprising of three proteins
produced by the organism viz. Protective antigen
,lethal factor and edema factor. The protective
antigen attach to the cell surface and they bind to
one another in groups of seven forming a doughnut
shaped space called ‘prepore’. Either the lethal
factor or edema factor binds to the prepore and
enters into the cell through endocytosis resulting in
destruction of cells. The main effect of toxins is to
increase the vascular permeability, which leads to
shock.
Clinical signs: Clinical signs in animals differ by
the species, with ruminants being the most at risk.
The peracute form most often affects ruminants,
including cattle, sheep and goats. Sudden death may
be the only clinical sign, so careful attention should
be paid to the carcass. The toxins in Bacillus
anthracis prevent the blood from clotting so animals
will often have bloody discharge coming from
orifices, including the mouth, nostrils, eyes, ears,
vulva, and anus. The carcass will decompose fairly
rapidly leading to bloating, but rigor mortis will not
be complete. The course of an acute infection is
usually 1-3 days but may take up to 7 days and will
affect ruminants, as well as horses. An acute infection
may manifest with a brief period of high fever (up to
107° F), excitement initially followed by depression,
muscle tremors, staggering, dyspneoa, cardiac
distress, and disorientation prior to death. Sub acute
to chronic infections occur in less susceptible
species such as pigs, but is also seen in cattle,
horses, dogs and cats. The main symptoms are
pharyngeal and lingual edema with animals dying
from asphyxiation. Extensive localized subcutaneous
edema of the ventrum, including the neck, sternum,
and flank can also be seen. The carcass will
decompose fairly rapidly leading to bloating, but rigor
22
 mortis will not be complete. Treatment with antibiotics
can be successful if begun early in the course of
the disease. Penicillin is the drug of choice for
treating affected animals. Doxycycline and
Ciprofloxacin are also very effective against anthrax.
LABORATORY DIAGNOSIS:
1.Direct demonstration of the organisms in the blood
smears: Blood smears are prepared from peripheral
blood or oozing blood and stained with a special stain
polychrome methylene blue. Wrights or Giemsa
stains can also be used. When the blood smears
are stained with polychrome methylene blue,
organisms appear as large blue bacilli surrounded
by a purplish granular stained capsule (
McFadyeans’ reaction).
2. Ascoli test- a thermoprecipitation test used to
demonstrate the presence of antigen in tissues like
ear piece or muzzle piece using standard
hyperimmuneserum.
3. Animal inoculation test: Guinea pigs and mice
are highly susceptible. Guinea pigs are inoculated
subcutaneously with pathological material or pure
culture.Animal dies within 2-3 days with marked
inflammatory lesions at the site and extensive
gelatinous oedema in the subcutaneous tissues.
Organisms are demonstrated in large numbers in
local lesions / heart blood / spleen.
Vaccination: Annual vaccination of livestock in
endemic areas is recommended. The most widely
used vaccine is the Sterne-strain vaccine
(Anthrax spore vaccine) produced from the non
encapsulated Sterne strain. The live spores are
suspended in 50% glycerol saline. All the susceptible
animals should be vaccinated once in six months in
endemic areas.
Prevention and control: Anthrax is a notifiable
disease, if anthrax is suspected the veterinarian and
local health officials should be contacted. Do not
open the carcass to perform a necropsy due
to the potential for contamination and
exposure, it is best to burn or bury the
carcasses and all contaminated materials. The
animal anthrax vaccine can be used on susceptible
healthy livestock. Then decontaminate the soil and
contaminated materials with 5% quicklime
(anhydrous calcium oxide). Hydrogen peroxide,
peracetic acid or gluteraldehyde may be good
alternatives. Commercially available bleach or 0.5%
hypochlorite solution (a 1:10 dilution of household
bleach) may be used but it may be corrosive to some
surfaces.
ENTEROTOXAEMIA
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Enterotoxaemia is an acute toxaemia resulting
sudden death of sheep caused by the proliferation
of the gram-positive anaerobe, Clostridium
perfringens type D, in the small intestine and the
liberation of epsilon (å) toxin and causes severe
vascular damage. C. perfringens is a normal
intestinal inhabitant. Disease often follows upsets in
the gut flora, which can result from sudden changes
to a rich diet or continuous feeding of concentrates.
Rapid multiplication of the organism and production
of å –toxin. The effects of å –toxin on the CNS and
other tissues cause sudden death, preceded in some
cases by clinical signs such as opisthotonus and
convulsions.
Transmission can occur by fecal-oral route, or by
ingestion of a large quantity of the bacteria through
contaminated soil, water or feed. In healthy animals,
most of the ingested C. perfringens type D are
destroyed in the rumen and abomasum. Although
the alkaline pH of the duodenum is quite favourable
for multiplication of these bacteria, toxaemia does
not occur, as continual movement of ingesta keeps
the bacterial population and toxin contents low.
Animals with high levels of epsilon toxin may move
about without showing signs of illness until found
dead or exhibiting the acute form of enterotoxaemia
.
Susceptible animals: The disease is prevalent in
sheep and goats, with per acute cases occurring at
3-10 weeks of age , although both acute and chronic
enterotoxaemia can occur in both young and adult
sheep and goats. The tendency for chronic cases
to occur is relatively higher in vaccinated adult goats,
while acute enterotoxaemia usually occurs in
unvaccinated young and adult goats.
The epsilon toxin is produced as an inactive
protoxin initially and activated by trypsin where in
trypsin removes a 13-residue N-terminal peptide.
Epsilon toxin is a potent toxin responsible for a rapidly
fatal enterotoxemia in sheep. One of the main
properties of epsilon toxin is the production of edema
especially in brain, necrosis of brain tissue and
death. The toxin is known to increase intestinal
permeability, and can also cause liver damage,
elevate blood pressure and cause an increase in
vascular permeability. This can lead to vascular
damage and edema in many organs including brain,
heart, lung and kidneys.
This disease is also known as lamb ‘overeating
disease’ or Pulpy kidney diseases. The organism
establishes in the gut and multiplies. The epsilon
toxin produces a systemic toxemia. This leads to CNS
lesions including opisthotonus, convulsions and
23
 sudden death. Kidney lesions are also commonly
associated with this form of the disease.
LABORATORY DIAGNOSIS: It requires evaluation
of clinical signs, gross and microscopic lesions,
bacteriologic culturing of appropriate specimens
(feces, intestinal contents) and typing of isolates.
Demonstration of toxins in the intestinal contents,
feces or serum (trypsin treated or untreated, neat
or mixed with antitoxins) are examined in
mice(injected intravenously) or in guinea
pigs(injected intra dermaly)
PCR genotyping can be useful complement to other
diagnostic methods. Genes for the major toxins
(epsilon toxin) can be detected.
Control: Formaldehyde Inactivated alum-
precipitated vaccine is widely used in the country
with satisfactory results. Biannual vaccination of all
adult and young sheep of above 3months old should
be followed in endemic areas .
HAEMORRHAGIC SEPTICAEMIA (SEPTICAEMIC
PASTEURELLOSIS/ BARBONE)
The disease is caused by Pasteurella multocida
type1 or B (6:B) and occasionally by type 4 (D) and
type E (1&2) (6:E in Africa) and is characterized by
per acute septicemia and high mortality rate.
Epidemiology: The disease is seen in cattle, buffalo,
sheep, goats, pigs, yaks, bisons, camels and horses.
It occurs in Asian countries, Europe, Russia and
Africa. Most affected animals are those exposed to
chilly and inclement weather or exhausted by heavy
work. Animals of all ages and breeds are susceptible
but most susceptible age group is 6 months to 2
years. Both morbidity and mortality rates vary from
50 -100 per cent. Mortality depends on the
immune status of the herd either acquired naturally
or by vaccinations.
After the outbreaks the causative agent persists on
the tonsillar and naso-pharyngeal mucosae of carrier
animals and as a commensal in healthy animals.
Spread occurs by ingestion of contaminated food,
carriers, clinical cases, ticks and insects. The saliva
of infected animals contains large numbers of
pasteurellae but the organisms do not survive on
pasture for more than 24 hours.
Pathogenesis: The portal of entry of infection is
tonsils. The organisms multiply freely in tissues,
respiratory tract, heart and gastro-intestinal tract and
results in severe septicaemia. Death occurs within
24 hours from an overwhelming endotoxaemia
resulting in extensive intra-vascular coagulation in
tissues, petechial and ecchimotic haemorrhages on
LIVESTOCK LINE, MARCH 2012
epicardium and tissues with yellowish gelatinous
exudates.
Clinical findings: Acute septicaemia
characterized by sudden onset of fever (106o-107o
F), profuse salivation, sub-mucosal petechiation,
severe depression, death resulting in 24 hours.
Animals may be found dead without any clinical
signs. Localizations in subcutaneous tissue resulting
in warm, painful swellings around the throat, dewlap,
brisket, perineum and severe dyspnoea. In some
cases in later stages animals develop pulmonary or
alimentary involvement. Pasteurella can be isolated
from saliva and blood. The disease in pigs is similar
to that in cattle.
Necropsy findings: Generalized petechial
haemorrhages under serosae, oedema of lungs and
lymph nodes. Sub-cutaneous infiltrations of
gelatinous fluid in some lesions of early pneumonia
and haemorrhagic gastro-enteritis. In lungs
congestion, consolidation, prominent thickening of
inter lobular septa. Isolation of organisms is best
from heart blood and spleen.
LABORATORY DIAGNOSIS: 1. Direct Microscopic
examination of blood smears or smears from
exudates, tissues such as spleen, liver stained with
Methylene blue or Geimsa, appearance of
characteristic bipolar organisms confirms the
disease. 2. Isolation and identification of the
causative agent from blood or nasal swab within a
few hours of death. During clinical phase, blood or
nasal swabs are not reliable. 3. Long bones of
carcasses are used for culturing. 4. Samples of blood
are injected to mice which will die within 24-36 hours.
Organisms can be isolated from mice. 5. Smears
from saliva, blood and aspirated exudates from
swellings reveal organisms. 6. Rapid ELISA to
identify the serotype.
Control: 1. Killed vaccine with oil adjuvant is highly
effective when used prophylactically and also in the
face of an outbreak. Solid immunity for at least 12
months is conferred. But the disadvantages are
persistent subcutaneous swellings and anaphylactic
shock. 2. Live vaccine of serotype B:3, 4 has been
successful and is free of anaphylactic shock. It gives
protection up to one year. Freeze-drying is
necessary for large scale production which is very
expensive. 3. Inactivated alum-precipitated vaccine
is widely used in India and has given satisfactory
results. Zoo-sanitary measures by treating the
affected areas with disinfectants and proper disposal
of dead carcasses and maintenance hygienic
conditions.


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 Livestock Improvement Strategies in Northern
Hilly Regions of India
Dibyendu Chakraborty1, A K Das2, N Kumar1 and D Kumar1
Division of Animal Genetics and Breeding, SKUAST-J, R S Pura, Jammu-181 102
U
Being vast country India having different agro-
climatic regions. The life-style of the people is largely
influenced by the habitat. The main source of income
for the people is agriculture-based. But, still there
are some places in India where cultivation is not
possible. At those places Livestock raring is the
major source of income. Northern hilly region is one
such agro-climatic region. Northern hilly region
comprises of Jammu & Kashmir, Himachal Pradesh,
Uttarakhand and hilly part of Uttar Pradesh. Main
Livestock species of this region are cattle, buffalo,
sheep, goat and horse. Beside these pigs, donkey,
camel and yak are also available.
Cattle
Due to hilly regions the indigenous cattle breeds of
this region are of small size. The cattle breeds of
this region are Panwar breed (Small hill-type cattle
found in foot-hills of Pilibhit district of UP); Pahari
(local non-descriptive breed); Ladhakhi (Kashmir
type of Indian hill cattle) (Maule, 1990).
During the year 2006-07 the organized breedable
population coverage was reached to 30.53%
(17.58% through AI & 12.95% through natural
breeding) which was only 5% at the inception of
implementation of NPCBB in the state of Uttarakhand
(Annual Report, 2006-07 ULDB). Crossbreed
animals constitute only 6% of the total cattle
population in HP (Negi, 1994). Similarly in Uttarkhand,
the Central Indian Himalayas, crossbred cattle make
up less than 2% of the total cattle population
(Sherpa, 1997).
According to breeding policy of the State, inheritance
of exotic blood i.e. Jersey/Holstein is to be kept at
50% and remaining 50% inheritance will be
contributed by Pahari /Hilly cattle (Dept. of Animal
Husbandry HP, 2007). In J&K state the crossbreeding
of cattle is going with Jersey and HF. The exotic
inheritance of crossbred cattle is restricted to 50%
only. Some local breeds are facing extinction due to
different state livestock development policies. Pahari
breed was vanishing as the department was
promoting the Jersey, Holstein and Red Sindhi
breeds in the state under its livestock development
1
Assistant Prof. , Div.- AGB, FVSc & AH, SKUAST-J, R S Pura, Jammu-181 102. (E-mail: dibyendu_vet40@yahoo.co.in)
2
Associate Professor, Div.- AGB, FVSc & AH, SKUAST-J, R S Pura, Jammu-181 102
LIVESTOCK LINE, MARCH 2012
policy (Chauhan, Mar. 24, 2009, Tribune news
service). Under bull production program of NPCBB
the ULDB has established one state of art Embryo
Transfer Technology Laboratory at Animal Breeding
Farm, Kalsi for the conservation & propagation of
Red Sindhi breed of cattle.
Buffalo
In the mountain villages, buffalo milk contributes 98%
of total milk (Singh, 1992). Livestock population trend
in the Central Himalayan Uttrakhand hills of India
shows that cattle population has declined by 5%
while buffalo population has increased by 15%
between 1978 and 1988 (Mehta, 1997). In Himachal
Pradesh buffalo breeding was initiated in 1980s. Due
to the lack of technical knowledge (like how in the
preservation of buffalo semen) breeding was
performed through natural services by locating 92
Murrah bulls in 1980 at different places in the state.
Thereafter through artificial insemination facilities
that had been extended to 190 extension stations
(Dept. of Animal Husbandry Himachal Pradesh, http:/
/www.ahdhp.org). Upgradation of local buffalo breeds
by using frozen semen of Murrah buffaloes is
practiced in J&K state.
Sheep
This region is well known for good quality sheep
breeds. Sheep breeds are extensively used for wool
production. The sheep breeds of this region are
Gaddi, Rampur Bushair, Bhakarwal, Poonchi,
Karnah, Gurez and Changthangi (Fig.1). Kashmir
Merino breed is originated from crosses of different
Merino types (at first Delaine Merinos, and
subsequently Rambouillet and Soviet Merinos) with
predominantly migratory native sheep breeds, such
as Gaddi, Bhakarwal and Poonchi. The level of
inheritance in the cross-bred animals included in
Kashmir Merino varies from very low to almost 100%
Merino. The level of 50 to 75% exotic inheritance
predominates (Handbook of Animal Husbandry,
2002).
According to the National Commission on Agriculture
(NCA, 1976) the breeding strategy in this region
involves breeding for apparel wool through cross-
32
 breeding indigenous breeds with exotic fine-wool
breeds. The indigenous breeds are improved
through selective breeding. Exotic breeds used for
cross breeding in this region are Rambouillet and
Merino. The native sheep breeds of J&K are facing
extinction due to large scale introduction of Merino
and Rambouillet (Nivsarkar et al., 1994). Sheep
crossbreeding with Polled Dorset (Mutton breed) has
remained confined to selected pockets in the Valley
such as Hajan block. Corriedale breed has shown
good adaptability and performance in the orchard
belt of Kashmir, i.e., Shopian area. Nowadays efforts
are made for crossbreeding with Garole sheep or
introducing of high fecundity gene (Fec B gene) for
production of twins and triplets to increase the sheep
population.
Goat
The goat breeds Bhakarwal, Changthangi, Chegu,
Gaddi, Kangan, White Himalayan are distributed in
J & K, H.P., Northern Punjab, Uttarakhand and Hill
districts of U.P (Fig. 2). Generally unrecognised
crossing with dairy goats with Angora is adopted by
HP and J & K states. No improvement programme of
Changthangi, Chegu and Gaddi breeds is still
initiated. Crossbreeding for pashmina fibre
production in Changthangi breed in J&K with exotic
breed Orenberg (Russia) is practiced (Source:
Handbook of Animal Husbandry, 2002). There are
over 150,000 pashima goats in Chanthang plateau
in Ladakh region, which contribute to 90 per cent of
pashima wool production in the country. (PTI,
February 20, 2009).
Ladakh has a large pool of genetic material within
its indigenous goat population and produces some
of the best pashmina fibre in the world. Thus, crosses
with foreign breeds have been discouraged.
Selective goat breeding is being conducted on
government farms to select for pure white hair so as
to raise the value of the pashmina and to improve
pashmina production from 200 g per goat to 300 g
by increasing secondary follicular hair density.
Lambing and kidding occurs in February-March, the
coldest months of the year, and mortality rates can
be as high as 40-70%. So the optimum breeding
time is from August to October so that green grass
is available for milking dams in April and May and
more milk will be available for newborns. Thus it will
reduce kid mortality. Reduced kid mortality from 30
to 2% and increased adult survivability by changing
goat breeding times on government goat farms were
reported (Development of Pashmina in Ladakh,
Jammu and Kashmir, 2009). Generally unrecognised
crossing with dairy goats with Angora was adopted
by HP and J & K. Crossbreeding for pashmina fibre
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production in Changthangi breed in J&K with exotic
breed Orenberg (Russia) is practised.
The CSK, Himachal Pradesh Agricultural University
has been sanctioned a new research project worth
Rs. 1.39 corer under the National Agricultural
Innovations Project (NAIP) of the Indian Council of
Agricultural Research (ICAR) to undertake research
and developmental activities for promoting pashmina
production and its efficient utilization in Himachal
Pradesh (February 11, 2009 http://
www.fibre2fashion.com/). Now a days cloning of
Pashmina goat is tried to increase the number of
Pashmina goat in Jammu and Kashmir state (http://
www.dnaindia.com/report.asp?newsid=1232619).
Horse
The Spiti ponies have two strains Spiti pure and
Konimare.
Spiti show by HP government and Zanskari show by
JK government helped in dissemination and
improvement of these breeds. In Zanskari horse
breeding farm at Padum Zanskar in Kargil district of
Ladak selective breeding is practiced for breed
improvement and conservation.
Donkey, yak and double hump camel
The modern domesticated asses have mainly
descended from the Nubian race. The greatest
contribution to animal husbandry that ass has made
is the production of mules. Characterization of yak
genetic resources was also considered, and a pilot
study was done by the National Bureau of Animal
Genetic Resources in the yak-raising areas (Pal,
2001). Doubled hump camel (Camelus bactrianus)
found in Nubra valley in Ladakh area of J & K.
Conclusions
Parasitic diseases cause a huge loss in Animal
Husbandry in Himachal Pradesh (Jithendran, 2000).
Inspite of efforts in animal breed improvement, the
bulk of livestock population in the Himalayan region
remain local species. There is paucity of feed and
fodder for the livestock, especially during winter
months as a result productivity suffers (A quarterly
news bulletin of SKUAST, Srinagar, January-march,
2007). So, attentions should be given to the Priority
production system- Pastoral systems. Due to large
introduction of exotic germplasm the indigenous
sheep and goat breeds of this region are facing
extinction. So, importance should be given on
selective breeding and sustainable management to
increase the productivity of these breeds as well as
conservation. Attentions should be given in the
following fields-
33
 v To develop an appropriate institutional framework ICIMOD (International Centre for Integrated Mountain
for the conservation, classification and utilization of Development), Kathmandu, Nepal.
animal genetic resources NCA, New Delhi. 1976 Report of the National
v Pasture management and additional feed Commission on Agriculture, Part VII: Animal
resources Husbandry. Govt. of India, Ministry of Agriculture and
v Disease control and herd management Irrigation, New Delhi.
v To promote regional gene banks for specific Negi G.C. 1994. Livestock development in Himachal
livestock species Pradesh: Retrospect and prospect. MFS Series 7.
ICIMOD (International Centre for Integrated Mountain
v To support and stimulate scientific and sustained
Development), Kathmandu, Nepal.
conservation work.
Pal, R. N. 2001. Yak husbandry in India. FAO
References
corporate document repository.
Domestic Animal Diversity Conservation &
Sherpa. 1997. Proceedings and recommendations
Sustainable Development, 2000.
of the SHERPA seminar on ‘Fodder Problems in the
Handbook of Animal Husbandry. 2002. Indian Council Himalayan Region of India’, held at Pashulok,
of Agricultural Research, New Delhi. Rishikesh, India, 22–23 December 1997.
http://ahdhp.org/achievements.html Singh V. 1992. Dynamics of unsustainability of
http://ahdhp.org/breeding.html mountain agriculture. Report of the MFS-ICIMOD
http://ahdhp.org/breeds_of_Sheep.html commissioned Study in the Garhwal Himalaya, India.
http://ahdhp.org/livestock.html ICIMOD (International Centre for Integrated Mountain
Development), Kathmandu, Nepal.
http://ahdhp.org/schemes.html
Fig 1: Sheep breeds in the northern temperate region of
http://gbpihed.gov.in/envis/HTML/vol82/vol82-geeta India
njali.html
h t t p : / / i n d i a . g o v. i n / g o v t / v i e w s c h e m e .
php?schemeid=945
http://news.webindia123.com/news/Articles/India/
20081019/1082519.html
http://nird.ap.nic.in/clic/solan.html
http://planningcommission.nic.in/plans/stateplan/
sdr_jandk/sdr_jkexecutive.pdf
http://planningcommission.nic.in/plans/stateplan/
sdr_jandk/sdr_jkch3a.pdf
http://www.dnaindia.com/report.asp?
newsid=1232619
h t t p : / / w w w. i f a d . o r g / l r k m / r e g i o n / p i / I C I M O D /
in_pashmina.html Fig. 2 Goat breeds in the northern temperate region of India
h t t p : / / w w w. i n d i a d a i r y. c o m / t e c h _ l i s t _
geniticsbreeding_sahiwal.html
http://www.mapsofindia.com/jammu-kashmir/
economy/animal-husbandry.html
h t t p : / / w w w. m e r i n e w s . c o m / c a t F u l l . j s p ?
articleID=15709858
http://www.skyscrapercity.com/archive/index.php/t-
488675.html
Jithendran, K.P. (2000). A brief profile of blood
protista of domestic animals in Himachal Pradesh.
Himachal Vet. J., 4: 19-21.
Mehta D.S. 1997. Development experiences and
options in a hill region: The case of Uttarakhand,
U.P., India. Discussion Paper Series MEI 97/4. (http://www.fao.org/docrep/004/X6532E/X6532E06.htm)

LIVESTOCK LINE, MARCH 2012 34

 IMPORTANCE OF BIOTECHNOLOGY IN ANIMAL FEEDING
Dharmendra Vyas And Ruchi Tripathi
Apollo College of veterinary medicine Agra road Jaipur
U
Biotechnology is the application of technologies,
such as recombinant DNA techniques, biochemistry,
molecular and cell biology, genetics and genetic
engineering, and cell fusion techniques etc. Using
living organisms or its products, to manufacture
industrial products including antibiotics, insulin, and
interferon, to improve plants or animals, to develop
microorganisms for specific uses, to identify targets
for pharmaceutical development, to transform
biological systems into useful processes and
products or to develop organisms for specific uses.
The largest impact of biotechnology on livestock
production is increasing the livestock feeds through
improving nutrient content as well as the digestibility
of low quality feeds through use of efficient feed
additives.
Its attention in two areas,
01) Development of genetically modified feed
ingredients in order to nutritionally enhance and
improve the production capabilities.
02) Improve certain feed ingredients which have
inherently low nutritional capabilities like high fiber,
anti- nutritive factors, low protein, and deficiency of
certain amino acids through the addition of feed
additives
Some of the limitations which the nutritionist face
during feed formulation are the antinutritive factors
like trypsin inhibitors, saponins, tannins, phytates,
oxalates, high fiber, limitation of phosphorus content
etc in feed. Developing genetically modified feed
having improved nutritional values could solve these
problems.
Value added feed stuff are Low phytate corn, High
oil corn, Low oligosaccharide soybean, Soybeans
with high lysine, GM crops with improved amino acid
profiles
Low phytate corn: All plant feed ingredients contain
natural phosphorus, which is only 30 % available,
and the rest 70 % is in the form of phytate
phosphorus. Grains with low phytate phosphorus and
high available phosphorus were made High oil corn.
This variety contains 87 % higher crude oil fat and
3.3% higher crude protein compared to typical corn.
Feeding studies with high oil corn on broilers shows
-Significant improvement in body weight & feed
LIVESTOCK LINE, MARCH 2012
conversion. -Less abdominal fat -Better feed to egg
ratio. -Egg yolks contained increased levels of
linoleic acid and oleic acid.
Low oligosaccharide soybean-: Soybeans contain
raffinose and stachyose the oligosaccharides, which
act as antinutritive factors. Genetically modified
soybeans with low oligosaccharides gave an
increased 3% in amino acid digestibility and 5 %
increased in dry matter digestibility. Soybeans with
high lysine: Increased lysine content from 3 % to 4.5
% It reduce the supplemental addition of lysine in
diets. GM crops with improved amino acid profiles
Great potential to decrease nitrogen excretion in
poultry.
Feed additives-: Adding specific nutrients to feed
improves animal digestion and thereby reduces feed
costs. A lot of feed additives are being currently used
and new concepts are continuously developed.
Enzymes Pro-biotic Pre-biotic Dietary amino acids
Toxin Binders Minerals and Vitamins Bypass proteins
Metabolic modifiers
Enzymes-: are biological catalysts and they improve
the nutrient availability from feedstuffs, lower feed
costs and reduce out put of waste into the
environment. 01) Microbial phytase as the result of
biotechnology is an enzyme that breaks down the
indigestible phytic acid (phytate) in cereals and
oilseeds and releases digestible phosphorus. This
reduces the use of expensive supplemental inorganic
phosphorus like dicalcium phosphate. Phytase also
releases minerals (Ca, Mg, Zn and K), amino acids
and proteins, which are complied with the Phytate
molecule.
02) Concentrate feed pellets could be made by
incorporating cellulase,pectinace and xylanase with
straw, bagasse and other agricultural waste.
Probiotics-: Probiotics can help to build up the
beneficial bacteria in the intestine and competitively
exclude the pathogenic bacteria. These bacteria
also release enzymes, which help in the digestion of
feed.
The common organisms in probiotic products are
Aspergillus oryzae, Lactobacillus acidophilus, L.
bulgaricus, L.plantarium, Bifidobacterium bifidium,
Streptococcus lactis and Saccharomyces cerevisiae.
35
 Can be administered through water or incorporated
in the feed. Useful in the early stages of chick growth
since the gut of the newly hatched chick is sterile
Helps to build up beneficial bacteria much faster than
the normal course.
Prebiotics-: Some of the prebiotics, which are
currently used in animal feed, are Mannan-
oligosaccharides (MOS), fructo-oligosaccharide and
mixed oligo-dextran. Mannan-oligosaccharides are
mainly obtained from cell walls of yeasts. Other
sources of MOS are copra or palm kernel meal. The
advantages of prebiotics are that it can stand high
palletizing temperatures in the feed and also have a
long shelf life.
Amino acids-: Essential amino acids are added as
supplement to the feed to get a balanced amino acid
profile. The new trend is to formulate diets on
digestible amino acid levels thereby reducing the
requirement of protein. Lysine is produced by
microbial fermentation and methionine is chemically
synthesized to add as supplement . Genetically
enhanced micro-organisms are being used to
produce threonine and tryptophan on a commercial
basis. Using all these amino acids it is possible to
lower dietary crude protein level by 2 – 3 %, which is
a substantial saving for the farmer..
Toxin Binders Present day methods are generally
use of organic acids and their salts like propionic
acid or adsorbents like bentonites, zeolites, hydroxyl
aluminosilicates. In the future, biotechnology based
products like microbes, herbal extracts or esterified
glucomannan could be used. Aqua extracts of garlic,
onion, turmeric, neem have been shown to exert
antifungal activity or inhibit aflatoxin production.
Minerals and Vitamins-: The absorption and
availability of inorganic trace minerals varies
depending upon the nature of the minerals (sulphate,
oxide or carbonate), their solubility, ionization etc.
Trace minerals are now being attached to
oligopeptides to make them more bioavailable.
Commercial preparations of proteinated selenium
and chromium are used in poultry. In the case of
vitamins due to varying availability and stability of
vitamins in ingredients supplemental vitamins are
incorporated in diets. These vitamins are much more
stable than naturally occurring forms.
Metabolic modifiers-: Metabolic modifiers are a
group of compounds that modify animal metabolism
in specific and directed ways. They have the overall
effect of Improving productive efficiency (weight gain
LIVESTOCK LINE, MARCH 2012
or milk yield per feed unit) Improving carcass
composition (lean:fat ratio) in growing animals,
Increasing milk yield in lactating animals and
Decreasing animal waste per production unit. Two
classes of compounds have received major focus,
1. Somatotropins (STS) and 2. Adrenergic agonists.
Commercially these compounds are produced by the
use of recombinant DNA technology to selectively
produce specific components for a species The most
common somatotropin is the bovine somatotropin
(bST) which is administered to dairy cows. Similarly
somatotropins have also been used in pigs which
resulted in greater nutrient use. Administering of â
adrenergic agonist components lead to improved
feed conversion ratio, daily weight gain and carcass
leanness. These components induce changes in
endocrine and cellular mechanism.
Merits and demerits-: Increase the digestibility of
low quality feeds through use of efficient feed
additives. Developing genetically modified grains with
improved nutritional values avoid limitations of
antinutritive factors like trypsin inhibitors, saponins,
tannins, phytates, oxalates and high fiber content
and limitation of phosphorus content during feed
formulation Less phosphorus would be thrown in the
litter and manure, which would lead to the control of
eutrophication.
If phytase enzyme used in poultry, aquaculture
integrated farmers are benefiting in controlling the
algal bloom due to the reduction in ground water
phosphorus levels. Also it reduces the use of
expensive supplemental inorganic phosphorus such
as dicalcium phosphate. Reduce the cost of animal
feed. Increased milk yields, improved productive
efficiency (milk/feed) and decreased animal waste.
The inclusion of genetically modified feedstuffs in
animal feed could also pose certain risks. GM plants
are produced by transferring foreign genes of
particular characteristics into feed grain crops. For
example introducing antibiotic resistant marker
genes may render common infectious diseases
untreatable or certain proteins may cause allergic
reactions to animals and humans. Hence proper lab,
field assessments as well as health assessments
have to be made before release of such plants for
commercial cultivation.
Due to enzymes attract import duty, making their
usage expensive. There are very few companies
producing enzymes due to lack of technology.


36
 Modifications of Polymerase Chain Reaction and
their Applications: An Overview
Arunkumar Patel1, Satish Kumar*, Pradip Ranaware1, Manish Kumar2
INTRODUCTION
U
Polymerase Chain Reaction (PCR) is in vitro an
enzymatic amplification of specific DNA sequences
in exponential manners. Kary Mullis (1983) is credited
for the invention of PCR assay. In general PCR
method require suitable DNA polymerase which is
able to withstand the high temperatures of >90°C
(>195°F) required for separation of the two DNA
strands in the DNA double helix after each replication
cycle. The DNA polymerases initially employed for
this was unable to withstand such high temperatures.
So the early procedures for DNA replication were
very inefficient, time consuming, and required large
amounts of DNA polymerase and continual handling
throughout the process. Therefore in each cycle it
needs incorporation of fresh polymerase which it
tedious in addition to time consuming. The discovery
of Taq polymerase (a DNA polymerase purified from
the thermophilic bacterium, Thermus aquaticus,
which naturally occurs in hot spring environments)
paved the way for dramatic improvements and thus
wide acceptance of the PCR method. The DNA
polymerase isolated from T. aquaticus is stable at
high temperatures remaining active even after DNA
denaturation, thus obviating the need to add new
DNA polymerase after each cycle. This allowed an
automated thermocycler-based process for DNA
amplification.
MODIFICATIONS OF PCR
On basis of need various modifications were
employed in basic PCR methods from time to time
which can be briefly describe as follow.
I. Basic modifications
Often only a small modification needs to be made to
the standard PCR protocol to achieve a desired goal.
1. Competitive PCRs
This is a method used for quantifying DNA using real-
time PCR. A competitor internal standard is co-
amplified with the target DNA and the target is
quantified from the melting curves of the target and
the competitor.
*Corresponding author, M.V.Sc., Division of Livestock Products Technology, Indian Veterinary Research Institute, Izatnagar, Bareilly,
UP -243 122, India., Phone: +91-9457917989 Email: leosatishkumar@gmail.com
1- Ph.D. Scholar, Division of Virology, Indian Veterinary Research Institute, Izatnagar, Bareilly, UP -243 122, India.
2- Ph.D. Scholar, Division of Physiology and Climatology, Indian Veterinary Research Institute, Izatnagar, Bareilly, UP -243122,India.
LIVESTOCK LINE, MARCH 2012
2. Long –PCR (LA-PCR)
It is used for the amplification of long target DNA
sequences. The key to LA-PCR is an enzyme, a
thermostable DNA polymerase, which possesses 3’
to 5’ exonuclease activity, or proofreading activity.
The efficiency drastically declines when incorrect
bases are incorporated. The 3’to5’ exonuclease
activity removes these misincorporated bases and
makes the further reaction proceed smoothly.
Therefore, the amplification of long DNA fragment
can be achieved. Enzymes like pfu were employed
for such PCR.
3. Multiplex PCR
Multiplex PCR is the term used when more the one
pair of primers is used in a PCR. The goal of Multiplex
PCR is to amplify several segment of target DNA
simultaneously. This PCR technique is used for
genetic screening, micro-satellite analysis and other
applications where it is necessary to amplify several
products in a single reaction. This technique often
requires extensive optimization because having
multiple primers pairs in a single reaction increases
the likelihood of primer-dimers and other non-specific
product that may interfere with the amplification of
specific products. In addition, the concentration of
individual primer pair often needs to be amplified
with differing efficiencies and multiple primer pair can
compete with each other in reaction. When two pair
of primers is used to amplify two segment of target
DNA simultaneously it is called as DUPLEX PCR (e.g.,
HA and NA gene segment of orthomyxoviruses).
When three pair of primers is used to amplify three
segment of target DNA simultaneously it is called as
TRIPLEX PCR (e.g., H, N, P gene segment of PPR
virus)
Advantages: Multiplex PCR has the potential to
produce considerable savings of time and effort
within the laboratory without compromising test utility.
Since its introduction, multiplex PCR has been
successfully applied in many areas of nucleic acid
diagnostics, including gene deletion analysis
37
 mutation and polymorphism analysis quantitative
analysis, and RNA detection.
Disadvantages: The optimization of multiplex PCRs
can pose several difficulties, including poor sensitivity
or specificity and/or preferential amplification of
certain specific targets. The presence of more than
one primer pair in the multiplex PCR increases the
chance of obtaining spurious amplification products,
primarily because of the formation of primer dimers.
These nonspecific products may be amplified more
efficiently than the desired target, consuming
reaction components and producing impaired rates
of annealing and extension. Thus, the optimization
of multiplex PCR should aim to minimize or reduce
such nonspecific interactions.
4. Multiplex Ligation-dependent Probe
Amplification (MLPA)
It permits multiple targets amplification with only a
single primer pair, thus avoiding the resolution
limitations of multiplex PCR.
5. Multiplex RT–PCR: (also known as Relative
RT-PCR)
It is commonly used for the semi-quantitative analysis
of gene expression levels. Typically, multiplex RT-
PCR is performed to determine the changes in
expression levels of gene in a series of tissue types
throughout stages of development or cellular
differentiation, or after specific experimental
treatment. Multiplex RT- PCR is also commonly used
to examine the expression patterns of a series of
related genes and to look at various regions of a
large message for mutation analysis.
6. Nested PCR
Nested PCR is used to increases the specificity of
DNA amplification, by reducing background due to
non-specific amplification of DNA. Two sets of primers
are being used in two successive PCRs. In the first
reaction, one pair of primers is used to generate
DNA products, which besides the intended target,
may still consist of non-specifically amplified DNA
fragments. The product(s) are then used in a second
PCR with a set of primers whose binding sites is fall
inside the amplicon of first primer set. Nested PCR
is often more successful in specifically amplifying long
DNA fragments than conventional PCR and so it has
greater specificity. Nested PCR reduce the
contamination in product due to amplification of
unexpected primer binding sites. When either primer
of primary set were employed with corresponding
new primer for secondary amplicon is known as semi-
LIVESTOCK LINE, MARCH 2012
nested PCR and when cDNA that has been reverse
transcribed from RNA before nested PCR then it is
known as Nested RT-PCR.
7. Quantitative PCR (Q-PCR)
It is used to measure the quantity of a PCR product
(preferably real-time). It is the method of choice to
quantitatively measure starting amounts of DNA,
cDNA or RNA. Q-PCR is commonly used to determine
whether a DNA sequence is present in a sample and
the number of its copies in the sample. The method
with currently the highest level of accuracy is
Quantitative real-time PCR. It is often confusingly
known as RT-PCR (Real Time PCR) or RQ-PCR.
QRT-PCR or RTQ-PCR is more appropriate
contractions. RT-PCR commonly refers to reverse
transcription PCR which is often used in conjunction
with Q-PCR. The commonly used methods for Q-
PCR are use of fluorescent dyes, such as SYBR
Green and fluorophore-containing DNA probes, such
as TaqMan Probe, Molecular Beacons, Scorpion
Probes etc.
Type of real-time PCR includes:
a. Real-time RT –PCR: This term refers to a real-
time PCR that is initiated with cDNA that has been
reverse transcribed from RNA.
b. Real-time RT-asymmetric PCR: This term refer
to an asymmetric PCR that is initiated with cDNA that
has been reverse transcribed from RNA.
c. Real-time RT- semi-nested PCR: This term refer
to a semi-nested PCR that is initiated with cDNA that
has been reverse transcribed from RNA.
d. Real-time RT-nested multiplex PCR: This term
refer to a nested PCR that is initiated with cDNA that
has been reverse transcribed from RNA and includes
multiple primer pairs at one or both of the consecutive
PCRs.
II. Pretreatments and extensions modifications
1. Colony PCR
Colony PCR is useful to differentiate the recombinant
colonies from non recombinant colonies. Primers for
the specific sequences (antibiotic resistance or
primers flanking a cloned region, specific gene
primer) should be used when preparing reaction
mixture and it allow rapid detection of transformants
containing the desired sequence.
2. Hairpin PCR
It is a method of error-free DNA amplification for
mutation detection. In this method sequence was first
38
 convert hairpin like structure. On amplification true
mutations will maintain the hairpin structure while
PCR error will disrupt the hairpin structure.
3. Hot-start PCR
This is a technique that reduces non-specific
amplification during the initial set up stages of the
PCR. The technique may be performed manually by
heating the reaction components to the melting
temperature (e.g., 95ÚC) before adding the
polymerase or specialized enzyme systems have
been developed that inhibit the polymerase’s activity
at ambient temperature, either by the binding of an
antibody or by the presence of covalently bound
inhibitors that only dissociate after a high-
temperature activation step. Hot-start/cold-finish
PCR is achieved with new hybrid polymerases that
are inactive at ambient temperature and are instantly
activated at elongation temperature. Now a day’s
various real time PCR pre mix available with follow
hot start procedures.
4. In-Situ PCR (IS-PCR)
IS-PCR is performed on fixed cells. DNA or RNA is
immobilized in their sub-cellular locations. In-Situ
Hybridization (ISH) or IS-PCR has proven to be a
very important molecular tool in diagnostic and
research and has significantly advanced the study
of gene structure and expression at the level of
individual cells .This technique has result in an
increased understanding of infectious and neoplastic
diseases. More recently, an intracellular reverse
transcription step to generate complimentary DNA
from mRNA template prior to in-situ PCR has been
for detection of low copy mRNA sequences. This
modification of in-situ PCR has been termed as “in-
situ RT-PCR” or “RT in-situ PCR” or “in-situ cDNA
PCR”. Utility of immuno-histochemistry, in-Situ
Hybridization and in-situ PCR amplification in the
surgical and cytopathology of viral infection has been
reported. .Main advantages are its low background,
high specificity, fast assay with shorter turn-around
time and no need of radioactive chemicals.
5. Ligation-mediated PCR
6. This method uses small DNA linkers ligated to the
DNA of interest and multiple primers annealing to
the DNA linkers; it has been used for DNA
sequencing, genome walking, and DNA footprinting.
7. Methylation-specific PCR (MSP)
The MSP method was used to detect methylation of
CpG islands in genomic DNA. DNA is first treated
with sodium bisulfite, which converts unmethylated
LIVESTOCK LINE, MARCH 2012
cytosine bases to uracil, which is recognized by PCR
primers as thymine. Two PCRs are then carried out
on the modified DNA, using primer sets identical
except at any CpG islands within the primer
sequences. At these points, one primer set
recognizes DNA with cytosines to amplify methylated
DNA, and one set recognizes DNA with uracil or
thymine to amplify unmethylated DNA. MSP using
qPCR can also be performed to obtain quantitative
rather than qualitative information about methylation.
8. RT-PCR (Reverse Transcription PCR)
RT-PCR is a method used to amplify, isolate or
identify a known sequence from a cellular or tissue
RNA. The PCR is preceded by a reaction using
reverse transcriptase to convert RNA to cDNA. RT-
PCR is widely used in expression profiling, to
determine the expression of a gene or to identify
the sequence of an RNA transcript, including
transcription start and termination sites and, if the
genomic DNA sequence of a gene is known, to map
the location of exons and introns in the gene. The 5'
end of a gene (corresponding to the transcription
start site) is typically identified by an RT-PCR method,
named RACE-PCR, short for Rapid Amplification of
cDNA Ends. AMV-RT and Mo-MLV-RT are commonly
used enzymes in RT –PCR.
9. Touchdown PCR
It is a variant of PCR in which degenerate primers
are used to reduce nonspecific background by
gradually lowering the annealing temperature as
PCR cycling progresses. The annealing temperature
at the initial cycles is usually a few degrees (3-5ÚC)
above the Tm of the primers used, while at the later
cycles, it is a few degrees (3-5ÚC) below the primer
Tm. The higher temperatures give greater specificity
for primer binding, and the lower temperatures permit
more efficient amplification from the specific products
formed during the initial cycle.
III. Primer modifications
1. Alu –PCR
This PCR is performed using the Alu primers
designed to have recognitions sequences of Alu
restriction enzyme.
2. Asymmetric PCR
A PCR in which the predominant product is a single
stranded DNA, as result of unequal primer
concentrations. It finds use in some types of
sequencing and hybridization probing where having
only one of the two complementary strands is
39
 required. PCR is carried out as usual, but with a great
excess of the primers for the chosen strand. Due to
the slow (arithmetic) amplification later in the reaction
after the limiting primer has been used up, extra
cycles of PCR are required.
3. LATE-PCR
It is modification of asymmetric PCR. LATE stands
for Linear-After-The-Exponential, LATE-PCR uses
a limiting primer with a higher melting temperature
(Tm) than the excess primer to maintain reaction
efficiency as the limiting primer concentration
decreasesmid-reaction.This type of PCR has been
used for the detection of a target gene of
Adenovirus.
4. Allele-specific PCR
This is a selective PCR amplification of one of the
alleles to detect single Nucleotide Polymorphism
(SNP). Selective amplification is usually achieved by
designing a primer such that the primer will match/
mismatch one of the alleles at 3’-end of the primer.
This diagnostic or cloning technique that requires
prior knowledge of a DNA sequence, including
differences between alleles, and uses primers whose
3' ends encompass the SNP. PCR amplification
under stringent conditions is much less efficient in
the presence of a mismatch between template and
primer, so successful amplification with an SNP-
specific primer signals presence of the specific SNP
in a sequence.
5. Assembly PCR or Polymerase Cycling
Assembly (PCA)
Assembly PCR is the artificial synthesis of long DNA
sequences by performing PCR on a pool of long
oligonucleotides with short overlapping segments.
The oligonucleotides alternate between sense and
antisense directions and the overlapping segments
determine the order of the PCR fragments thereby
selectively producing the final long DNA product.
6. Box –PCR
Box elements are repetitive sequences elements in
bacterial genome such as streptococcus genome.
Single PCRs targeting to the repeats can be used
to fingerprint bacterial species.
7. Consensus –PCR
This PCR is carried out with flanking primer to amplify
repeat regions from a number of species. In this case,
degenerate /consensus primers may be used for
amplified the flanking sequences.
LIVESTOCK LINE, MARCH 2012
8. Degenerate PCR
Degenerate PCR is in most respect identical to
ordinary PCR, but with one major difference i.e.
instead of using specific PCR primers with a given
sequence, mixed
PCR primers will be used .That is, “wobble” are
inserted into the primers in case if the exact sequence
of the gene is not known so that there will be more
than one possibility for exact amplification.
Degenerate PCR has proven to be a very powerful
tool to find “new” genes or gene families’. To design
primer conserved and variable parts is studied by
alignment of various sequences of related proteins.
9. Differential Display PCR (DD-PCR):
DD-PCR is used for cloning purpose; it combines
the comparative analysis of several samples with the
sensitivity of PCR. Recent studies shows that by
modifying the primer design, sampling of differentially
expressed genes can be greatly enhanced and
relevant genes can be isolated.
10. Miniprimer PCR
Miniprimer PCR uses a novel thermostable
polymerase (S-Tbr) that can extend from short
primers (“smalligos”) as short as 9 or 10 nucleotides,
instead of the approximately 20 nucleotides required
by other polymerase like Taq. This method permits
PCR targeting smaller primer binding regions, and
is particularly useful to amplify unknown, but
conserved, DNA sequences, such as the 16S (or
eukaryotic 18S) rRNA gene. 16S rRNA miniprimer
PCR was used to characterize a microbial mat
community growing in an extreme environment.
Miniprimer PCR may reveal new dimensions of
microbial diversity. By enlarging the “sequence
space” that may be queried by PCR primers, this
technique may enable novel PCR strategies that are
not possible within the limits of primer design imposed
by Taq and other commonly used enzymes.
11. Inverse PCR
It is a type of standard PCR that is used to amplify
the segment of DNA that lies between two inward –
pointing primers. Inverse PCR (also known as
inverted or inside out) is used to amplify and clone
unknown DNA that flanks one end of known DNA
sequence and for which no primers are available.
The technique involves digestion by a restriction
enzyme of a preparation of DNA containing the known
sequences and its flanking region. The individual
restriction fragments are converted into circles by
40
 intermolecular ligation and circularized DNA is then
used as template in PCR. The unknown sequence
is amplified by two primers that bind specifically to
the known sequence and point in opposite direction.
12. PCR-RFLP
Restriction Fragment Length Polymorphism (RFLP)
is a technique in which organism may be
differentiated by analysis of patterns derived from
cleavage of their DNA by a set of restriction enzyme
(RE). If two organisms differ in the distance between
sites of cleavage of a particular restriction
endonuclease, the length of the fragment produced
will differ when DNA is digested with a RE. The
similarity of the patters generated can be used to
differentiate species (and even strains) from one
another. By designing primers that will introduce or
destroy a restriction site for one of the alleles, the
PCR product for SNP alleles can be distinguished
by restriction fragment length analysis.
13. Overlap-extension PCR
It is a genetic engineering technique allowing the
construction of a DNA sequence with an alteration
inserted beyond the limit of the longest practical
primer length.
14. Vectorette PCR
Vectorette PCR is a method that enables the
amplification of specific DNA fragment in situation
where the sequence of only primer is known. Thus it
extends the application of PCR to stretches of DNA
where the sequence information is only available at
one end.
IV. Post PCR modifications
PCR-ELISA
The PCR product are labeled (digoxigenin) during
amplification. This labeled amplicon is immobilize in
immuno-well plate. Normal ELISA is then employed
to quantitate PCR product.
V. Modification in DNA Polymerases
There are several DNA polymerases that are used
in PCR viz.
1. Klenow fragment
The Klenow fragment, derived from the original DNA
Polymerase-I from E. coli, was the first enzyme used
in PCR. Because of its lack of stability at high
temperature, it needs be replenished during each
cycle, and therefore is not commonly used in PCR.
2. Bacteriophage T4 DNA polymerase
LIVESTOCK LINE, MARCH 2012
The bacteriophage T4 DNA polymerase was also
initially used in PCR. It has a higher fidelity of
replication than the Klenow fragment, but is also
destroyed by heat.
3. Taq polymerase
The DNA polymerase from Thermus aquaticus, was
the first thermostable polymerase used in PCR, and
is still the one most commonly used.
4. Stoffel fragment
The Stoffel fragment is made from a truncated gene
for Taq polymerase and expressed in E. coli. It is
lacking 5'-3' exonuclease activity, and may be able
to amplify longer targets than the native enzyme.
5. Faststart polymerase
Faststart polymerase is a variant of Taq polymerase
that requires strong heat activation, thereby avoiding
non-specific amplification due to polymerase activity
at low temperature. Currently using in most of real
time PCR.
6. Pfu DNA polymerase
Enzyme Pfu DNA polymerase, isolated from the
Pyrococcus furiosus, has proofreading activity, and
a 5-fold decrease in the error rate of replication
compared to Taq. Since errors increase as PCR
progresses, Pfu is the preferred polymerase when
products are to be individually cloned for sequencing
or expression.
7. Vent polymerase
Vent polymerase is an extremely thermostable
polymerase isolated from Thermococcus litoralis.
8. Tth polymerase
Tth polymerase is a thermostable polymerase
from Thermus thermophilus. It has reverse
transcriptase activity in the presence of Mn2+ions,
allowing PCR amplification from RNA targets.
VI. Other modifications
1. Amplified Fragment Length Polymorphisms
(ALFP)
The use of randomly amplified polymorphic DNA
(RAPD) markers in systematic studies has been
reviewed. The use of randomly amplified polymorphic
DNA (RAPD) markers in systematic levels of
polymorphism and their low cost compared to other
techniques, such as allozymes and Restricrion
Fragment length polymorphism (RFPL). Two new
marker methodologies appear to be supplanting
RAPD analysis (AFLPs and simple sequence
41
 Repeats).Whilst the RAPD technique is fairly simple,
both AFLP and SSR protocols are technically
demanding.
2. Forensic PCR
Forensic PCR is a PCR which was normally
employed in various vetro-legal cases to find the
source of suspected samples. The VNTR (variable
and frequently observed tandem repeats in human
individual Genome) locus is amplified by PCR to
compare DNA samples from different sources.
3. Helicase-dependent amplification
This technique is similar to traditional PCR, but uses
a constant temperature rather than cycling through
denaturation and annealing/extension cycles. DNA
Helicase, an enzyme that unwinds DNA, is used in
place of thermal denaturation, it is very well exploited
in LAMP system.
4. RACE-PCR
Rapid Amplification of cDNA Ends PCR (RACE-PCR)
is used to obtain the 3’ end of a cDNA; it requires
some sequence information internal to the mRNA
under study. The sequence information obtain from
technique can be utilized to obtained full length cDNA
clones using the 5’ RACE technique.
5. Solid Phase PCR
It encompasses multiple meanings, including colony
Amplification (where PCR colonies are derived in a
gel matrix, for example), ‘Bridge PCR’ (the only primers
present are covalently linked to solid support
surface), conventional Solid Phase PCR (where
Asymmetric PCR is applied in the presence of solid
support bearing primer with sequence matching one
of the aqueous primers) and Enhanced Solid Phase
PCR (where conventional Solid Phase PCR can be
improved by employing high Tm solid support primer
with application of a thermal ‘step’ to favor solid
support priming).
6. Universal Fast Walking
This method allows genome walking and genetic
fingerprinting using a more specific ‘two-sided’ PCR
than conventional ‘one-sided’ approaches (using
only one gene-specific primer and one general
primer - which can lead to artefactual ‘noise’) by virtue
of a mechanism involving lariat structure formation.
Streamlined derivatives of UFW are LaNe
RAGE(lariat-dependent nested PCR for rapid
amplification of genomic DNA ends), 5’RACE LaNe
and 3’RACE LaNe.
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APPLICATIONS OF PCR
I. Medical applications
PCR has been applied to a large number of medical
procedures:
1. Genetic Testing -The first application of PCR
was for genetic testing, where a sample of DNA is
analyzed for the presence of genetic disease
mutations. Prospective parents can be tested for
being genetic carriers, or their children might be
tested for actually being affected by a disease. DNA
samples for prenatal testing can be obtained by
amniocentesis, chorionic villus sampling, or even by
the analysis of rare fetal cells circulating in the
mother’s bloodstream. PCR analysis is also essential
to Preimplantation genetic diagnosis, where
individual cells of a developing embryo are tested
for mutations.
2. Sensitive Test For Tissue Typing- PCR can
also be used as part of a sensitive test for tissue
typing, vital to organ transplantation. As of 2008,
there is even a proposal to replace the traditional
antibody-based tests for blood type with PCR-based
tests.[2]
3. Oncogenes: Many forms of cancer involve
alterations to oncogenes. By using PCR-based tests
to study these mutations, therapy regimens can
sometimes be individually customized to a patient.
II. Infectious disease applications
Characterization and detection of infectious disease
organisms have been revolutionized by PCR:
1. Human Immunodeficiency Virus: The Human
Immunodeficiency Virus (or HIV), responsible for
AIDS, is a difficult target to find and eradicate. The
earliest tests for infection relied on the presence of
antibodies to the virus circulating in the bloodstream.
However, antibodies don’t appear until many weeks
after infection, maternal antibodies mask the infection
of a newborn, and therapeutic agents to fight the
infection don’t affect the antibodies. PCR tests have
been developed that can detect as little as one viral
genome among the DNA of over 50,000 host cells.[3]
Infections can be detected earlier, donated blood
can be screened directly for the virus, newborns can
be immediately tested for infection, and the effects
of antiviral treatments can be quantified.
2. Detection of Disease Organisms: Some
disease organisms, such as that for Tuberculosis,
are difficult to sample from patients and slow to be
grown in the laboratory. PCR-based tests have
42
 allowed detection of small numbers of disease
organisms (both live and dead), in convenient
samples. Detailed genetic analysis can also be used
to detect antibiotic resistance, allowing immediate
and effective therapy. The effects of therapy can
also be immediately evaluated.
3. Monitoring Spread of A Disease: The spread
of a disease organism through populations of
domestic or wild animals can be monitored by PCR
testing. In many cases, the appearance of new
virulent sub-types can be detected and monitored.
The sub-types of an organism that were responsible
for earlier epidemics can also be determined by PCR
analysis.
III. Forensic applications
The development of PCR-based genetic (or DNA)
fingerprinting protocols has seen widespread
application in forensics:
1.Genetic fingerprinting: In its most discriminating
form, Genetic fingerprinting can uniquely discriminate
any one person from the entire population of the
world. Minute samples of DNA can be isolated from
a crime scene, and compared to that from suspects,
or from a DNA database of earlier evidence or
convicts. Simpler versions of these tests are often
used to rapidly rule out suspects during a criminal
investigation. Evidence from decades-old crimes can
be tested, confirming or exonerating the people
originally convicted.
2. Parental testing: Less discriminating forms of
DNA fingerprinting can help in Parental testing, where
an individual is matched with their close relatives.
DNA from unidentified human remains can be tested,
and compared with that from possible parents,
siblings, or children. Similar testing can be used to
confirm the biological parents of an adopted (or
kidnapped) child. The actual biological father of a
newborn can also be confirmed (or ruled out).
IV. Research applications
PCR has been applied to many areas of research in
molecular genetics:
1. Rapid Production of DNA: PCR allows rapid
production of short pieces of DNA, even when
nothing more than the sequence of the two primers
is known. This ability of PCR augments many
methods, such as generating hybridization probes
for Southern or northern blot hybridization. PCR
supplies these techniques with large amounts of pure
DNA, sometimes as a single strand, enabling analysis
even from very small amounts of starting material.
LIVESTOCK LINE, MARCH 2012
2. Extract Segments From A Completely
Unknown Genome: The task of DNA sequencing
can also be assisted by PCR. Known segments of
DNA can easily be produced from a patient with a
genetic disease mutation. Modifications to the
amplification technique can extract segments from
a completely unknown genome, or can generate just
a single strand of an area of interest.
3. DNA Cloning: PCR has numerous applications
to the more traditional process of DNA cloning. It can
extract segments for insertion into a vector from a
larger genome, which may be only available in small
quantities. Using a single set of ‘vector primers’, it
can also analyze or extract fragments that have
already been inserted into vectors. Some alterations
to the PCR protocol can generate mutations (general
or site-directed) of an inserted fragment.
4. Human Genome Project: A Sequence-tagged
site is a process where PCR is used as an indicator
that a particular segment of a genome is present in
a particular clone. The Human Genome Project found
this application vital to mapping the cosmid clones
they were sequencing, and to coordinating the results
from different laboratories.
5. Phylogenic Analysis: An exciting application of
PCR is the phylogenic analysis of DNA from ancient
sources, such as that found in the recovered bones
of Neanderthals, or from frozen tissues of
Mammoths. In some cases the highly degraded DNA
from these sources might be reassembled during
the early stages of amplification.
6. Gene Expression: A common application of PCR
is the study of patterns of gene expression. Tissues
(or even individual cells) can be analyzed at different
stages to see which genes have become active, or
which have been switched off. This application can
also use Q-PCR to quantitate the actual levels of
expression
7. Genetic Mapping: The ability of PCR to
simultaneously amplify several loci from individual
sperm has greatly enhanced the more traditional
task of genetic mapping by studying chromosomal
crossovers after meiosis. Rare crossover events
between very close loci have been directly observed
by analyzing thousands of individual sperms.
Similarly, unusual deletions, insertions,
translocations, or inversions can be analyzed, all
without having to wait (or pay for) the long and
laborious processes of fertilization, embryogenesis,
etc.
43
 V. Others
PCR is also important in answering basic scientific
questions. In the field of evolutionary biology, PCR
has been used to establish relationships among
species. In anthropology, it has used to understand
ancient human migration patterns. In archaeology,
it has been used to help identify ancient human
remains. Paleontologists have used PCR to amplify
DNA from extinct insects preserved in amber for 20
million years. The Human Genome Project, which
had a goal of determining the sequence of the 3
billion base pairs in the human genome, relied
heavily on PCR. The genes responsible for a variety
of human diseases have been identified using
PCR. For example, a PCR technique called multiplex
PCR identifies a mutation in a gene in boys suffering
from Duchenne muscular dystrophy. PCR can also
be used to search for DNA from foreign organisms
such as viruses or bacteria.
CONCLUSION
Polymerase Chain reaction is one of the most
important molecular diagnostic tools. It is used in all
the fields of biology as a diagnostic because of its
high sensitivity and specificity. Theoretical
consideration and practical applications indicate that
PCR and RT-PCR assay system share several
advantage over other quantitative molecular
methodologies, thus suggesting that these technique
are the methods of choice for the absolute
quantitation of viral nucleic acid. The PCR is very
promising to elucidate the etiological agent of which
is present in too small numbers to be detected by
traditional techniques, agents difficult or impossible
to cultivate and in making distinction between
infection and rejection in transplant recipients. Thus
the advent of nucleic acid amplification techniques
for the clinical laboratory provides not only new
diagnostic opportunities but new challenges as well.
REFERENCES:
1. Bell J. The polymerase chain reaction. Immunol.
Today 1989; 10:351-4.
2. Boehnke M et.al. “Fine-structure genetic mapping
of human chromosomes using the polymerase chain
reaction on single sperm.” Am. J. Hum. Genet. Vol.
45(1) pp. 21-32 (1989).
3. Erlich HA, Gelfand DH, Saiki RK. Specific DNA
amplification. Nature 1988; 331:461-2.
4. Frohman MA, Dush MK, Martin GR. Rapid
production of fulllength cDNAs from rare transcripts:
amplification using a single gene-specific
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oligonucleotide primer. Proc. Natl. Acad. Sci. USA
1988; 85:8998-9002.
5. Giulietti A,Overbergh L , Valckx D: An overview of
real ime quantitation PCR: application to quantify
cytokine gene expression. Methods 2001;25: 386-
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6. Kwok S et.al. “Identification of HIV sequences by
using in vitro enzymatic amplification and oligomer
cleavage detection.” J. Virol. Vol. 61(5) pp. 1690-4
(1987).
7. Kwok S and Higuchi R. Avoiding false positives
with PCR. Nature 1989; 339:237-8.
8. Maitland N. Report from 1st National Symposium
on the Polymerase Chain Reaction, 9 May 1989,
London.
9. Marx JL. Multiplying genes by leaps and bounds.
Science 1988; 240: 1408-10.
10. Quill E “Blood-Matching Goes Genetic” Science
Magazine (14 March 2008) pp. 1478-1479.
11. Ririe KM; Rasmussen RP and Wittwer CT: product
differentiation by analysis of DNA melting curve
during polymerase chain reaction. Anal. Biochem.
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12. Saiki RK et.al., “Enzymatic Amplification of â-
globin Genomic Sequences and Restriction Site
Analysis for Diagnosis of Sickle Cell Anemia” Science
vol. 230 pp. 1350-54 (1985).
13. Saiki RK, Gelfand DH, Stoffel S, et.al, 1988..
Primer-directed enzymatic amplification of DNA with
a thermostable DNA polymerase. Science 1988;
239:487-91.
14. Schochetman G, Ou C-Y, Jones WK. 1988.
Polymerase chain reaction. J. Infect. Dis. 1988;
158:1154-7.
15. Triglia T, Peterson MG, Kemp DJ. 1988. A
procedure for in vitro amplification of DNA segments
that lie outside the boundaries of known sequences.
Nucleic Acids Res. 1988; 16:8186.
16. White TJ, Arnheim N, Erlich HA. 1989. The
polymerase chain reaction. Trends Genet. 1989;
5:185-9.
17. Wittwer C. 2001.Rapid cycle real time PCR:
Method s and application, In Rapid cycle real time
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,Heidelberg, Germany 2. Niesters H.G: Quantitation
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 Impact of nanotechnology in veterinary
science – a review
S. Ganguly1*, A. Prasad2, I. Paul3, D. Seth4, S.K. Mukhopadhayay5
Uwill revolutionize animal health and help to boost up
AbstractNanotechnology refers to the use of very tiny
(nano-scale) materials in a range of novel ways. ‘Nano’
means tiny and nano-particles are tiny particles, more
than 8000 times smaller than a human hair. The properties
of nano-particles make them suitable for a range of
environmental applications, both in terms of improving
existing environmental problems or by anticipating and
preventing future environmental problems. Some of the
greatest potential uses or application of nanotechnology
in the environment are as biosensors and in the sectors
of treatment, agriculture, veterinary science, fisheries,
bioremediation and for green nanotech manufacturing and
engineering. The present article has been constructed
considering the tremendous potential and application of
nanoscience and nanotechnology in the concerned
fields.Keywords: nanotechnology; nano-particles;
veterinary science; nanoscience.IntroductionVeterinary
health care is a highly responsible and growing concern
not only for pet owners, but also for our nation and
government. With an ever increasing pet population
throughout the globe, along with higher costs for
medications and veterinary care, the need for new
solutions is urgent. At this period of time the main
objectives of Veterinary Medicine is to excel according
to the accepted standards of scientific excellence in the
creation of new knowledge and its translation into improved
health for the other species with which we share our world,
to create more effective veterinary services and products
and to strengthen the veterinary education
system.Nanotechnology has a tremendous potential to
revolutionize agriculture and livestock sector. It can
provide new tools for molecular and cellular biology,
biotechnology, veterinary physiology, animal genetics,
reproduction etc. which will allow researchers to handle
biological materials such as DNA, proteins or cells in
minute quantities usually nano-liters or pico-liters.
Nanotechnology tools like micro-fluidics, nano-materials,
bio-analytical nano-sensors, etc. has the potential to solve
many more puzzles related to animal health, production,
reproduction and prevention and treatment of diseases.
It is reasonable to presume that in the upcoming year’s
nanotechnology research will reform the science and
technology of the animal health and will help to boost up
the livestock production. Nanotechnology will have a
profound impact, but not in the immediate future as it is
in the early stages of its development and needs to equip
scientists, engineers and biologists to work at the cellular
and molecular levels for significant benefits in healthcare
and animal medicine. But It is reasonable to presume
that in the upcoming year’s nanotechnology research
1,2
Department of Veterinary Microbiology, Faculty of Veterinary Science & Animal Husbandry, Birsa Agricultural University, Kanke,
Ranchi - 834 006 (Jharkhand), India. 3Department of Veterinary Microbiology, WBUAFS, India.
4,5
Department of Veterinary Pathology, Faculty of Veterinary & Animal Sciences, West Bengal University of Animal & Fishery
Sciences (WBUAFS), Kolkata - 700 037 (West Bengal), India. *Corresponding author, E-mail: ganguly38@gmail.com
LIVESTOCK LINE, MARCH 2012
livestock production (Patil et al., 2009).
Livestock and fisheries will be affected by the
nanotechnology revolution. While the great hopes of nano-
medicine are disease detection and new pharmaceuticals
for humans, veterinary applications of nanotechnology
may become the proving ground for untried and more
controversial techniques - from nano-capsule vaccines
to sex selection in breeding. Nanotechnology, dealing
with functional structures and materials smaller than
100nm, is emerging as a truly interdisciplinary research
area spanning several traditional scientific disciplines. In
keeping with the growing trend, there is a strong need for
a platform to share original research related to applications
of nanotechnology in biomedical fields.In the era of new
health related technologies, Veterinary Medicine will enter
a phase of new and incredible transformations. The major
contributor to those changes is our recent ability to
measure, manipulate and organize matter at the nano-
scale level. Our understanding of the principles that rule
the nano-scale world will be of great impact on veterinary
research leading to new discoveries never before
imagined.Nanotechnology has the potential to impact not
only the way we live, but also the way we practice
veterinary medicine. Today scientists foresee that the
progress in the field of nanotechnology could represent a
major breakthrough in addressing some of our technical
challenges not only in engineering but also in the fields
of both human and veterinary medicine. Very soon
engineers will develop tiny motors to power computers
and appliances and doctors will have miniature devices
that aim to fight cancer on the molecular level at their
disposal.In the veterinary community, some of the
principal areas of nanotechnology research are currently
being undertaken in the world of medicine because of
the vast scope of the medical applications of
nanotechnology. Many discoveries of veterinary and allied
professions in the field of nanotechnology have been
made till date and it is needed to provide a glimpse of the
potential important targets for nanotechnology in the field
of veterinary medicine. However, nanotechnology is in its
early stage of development and it may take several years
to perform the necessary research and conduct clinical
trials for obtaining meaningful results, but professionals
should begin to take note of it (Feneque, 2003).Biochips
- current and future industry applications Using
biochips, biological samples such as blood, tissue and
semen can be instantaneously analysed and
manipulated. In fewer than five years, biochips have
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 become a standard technology for genomics and drug
discovery and they are now moving into commercial
healthcare and food safety applications (ETC Group
Report, 2004). Use of biochips (Microarrays) to study
genetic sequencesA biochip (or microarray) is a device
typically made of hundreds or thousands of short strands
of artificial DNA deposited precisely on a silicon circuit.
In DNA arrays, each DNA strand acts as a selective probe
and when it binds to material in a sample (e.g. blood) an
electrical signal is recorded. Rather like conducting a
word search across a piece of text, the biochip is able to
report back on found genetic sequences based on the
DNA probes built into it. The best known biochips are
those produced by Affymetrix, the company that
pioneered the technology and was first to produce a DNA
chip that analyses an entire human genome on a single
chip the size of a dime (ETC Group Report, 2004). Using
biochips in biowarfare agents and in disease
detection applicationsIn addition to DNA biochips, there
are other variations that detect minute quantities of
proteins and chemicals in a sample, making them useful
for detecting bio-warfare agents or disease. Biochip
analysis machines the size of an inkjet printer are
commercially available from companies such as Agilent
(Hewlett-Packard) and Motorola - each able to process
up to 50 samples in around half an hour (ETC Group
Report, 2004). Using biochips for disease detection
in animals and for tracing the source of foodsChips
can be used for early disease detection in animals.
Researchers at the University of Pretoria are developing
biochips that will detect common diseases borne by ticks.
Biochips can also be used to trace the source of food
and feeds. For example, bioMérieux’s “FoodExpert-ID”
chip rapidly tests feed to detect the presence of animal
products from forty different species as a means to locate
the source of pathogens - a response to public health
threats such as avian flu and mad cow disease (ETC
Group Report, 2004). Use of biochips in animal
breeding to remove genetic diseasesOne goal is to
functionalise biochips for breeding purposes. With the
mapping of the human genome behind them, geneticists
are now rapidly sequencing the genomes of cattle, sheep,
poultry, pig and other livestock hoping to identify gene
sequences that relate to commercially valuable traits such
as disease resistance and leanness of meat. By including
probes for these traits on biochips, breeders will be able
to speedily identify champion breeders and screen out
genetic diseases (ETC Group Report, 2004).
‘Microfluidics’ and ‘Nanofluidics’Microfluidics is a
newer technology platform on the same scale as biochips.
Microfluidic and nanofluidic systems analyse by
controlling the flow of liquids or gases through a series of
tiny channels and valves, thereby sorting them, much as
a computer circuit sorts data through wires and logic
gates. Microfluidic channels, often etched into silicon,
can be less than 100 nm wide. This allows them to handle
biological materials such as DNA, proteins or cells in
minute quantities - usually nano-liters or pico-liters (1000
times smaller than a nano-liter). Microfluidics not only
enable very precise analysis, they also open up the
potential for manipulation of living matter by mixing,
separating and handling different components at the nano-
scale (ETC Group Report, 2004). Use of microfluidics
in livestock breedingMicrofluidics is being used in
livestock breeding to physically sort sperm and eggs.
Leader in this field is XY, Inc. of Colorado (USA), which
is using a microfluidic technique called flow cytometry to
segregate male and female sperm for sex selection. XY
has successfully bred sex-selected horses, cattle, sheep
LIVESTOCK LINE, MARCH 2012
and pigs and now provides its technology to commercial
breeders. Nanotech startup Arryx, which has developed
a new microfluidic system called MatRyx, uses a
nanotechnique in which tiny laser tractor beams trap
individual sperm and then sort them by weight. MatRyx
can sort around 3,000 sperm per second, and aims for
commercialisation in cattle breeding. “This way dairy
farmers can have cows and beef farmers can have bulls
that have more meat,” was explained by Arryx’s CEO,
Lewis Gruber, with a goal to produce a simple one-button
sex sorter (ETC Group Report, 2004). Uses of
microfluidic devices in biomimeticsMatthew Wheeler,
University of Illinois professor of animal science, has gone
one further in developing a microfluidic device that not
only sorts sperm and eggs but also brings them together
in a way that mimics the movement of natural reproduction
and then handles the resulting embryo. According to Dr.
Wheeler, such a technique would make mass production
of embryos cheap, quick and reliable. He and his
colleagues have started a spin-off company, Vitaelle, to
commercialise this technology (ETC Group Report, 2004).
Nanotechnology, as an enabling technology, has the
potential to revolutionize veterinary medicine. Examples
of potential applications in animal, agriculture and
veterinary medicine include disease diagnosis and
treatment delivery systems, new tools for molecular and
cellular breeding, identity preservation of animal history
from birth to a consumer’s table, the security of animal
food products, major impact on animal nutrition scenarios
ranging from the diet to nutrient uptake and utilization,
modification of animal waste as expelled from the animal,
pathogen detection and many more. Existing research
has demonstrated the feasibility of introducing nano-
shells and nano-tubes into animals to seek and destroy
targeted cells. Thus, building blocks do exist and are
expected to be integrated into systems over the next
couple of decades on a commercial basis. While it is
reasonable to presume that nano-biotechnology industries
and unique developments will revolutionize veterinary
medicine in the future, there is a huge concern, among
some persons and organizations, about food safety and
health as well as social and ethical issues which can
delay or derail technological advancements (Scott, 2007).
The U.S. Food and Drug Administration (FDA) regulates
a wide range of products, including foods, cosmetics,
drugs, devices, veterinary products, and tobacco products
some of which may utilize nanotechnology or contain
nanomaterials. Nanotechnology allows scientists to
create, explore and manipulate materials measured in
nanometers (billionths of a meter). Such materials can
have chemical, physical, and biological properties that
differ from those of their larger counterparts.
References
ETC Group (Action Group on Erosion, Technology and
Concentration) Report (November 2004) ‘Down on the
Farm: the Impact of Nano-Scale Technologies on Food
and Agriculture’
Jose Feneque (December 2003) Brief Introduction To The
Veterinary Applications Of Nanotechnology,
Nanotechnology Now
N.R. Scott (2007) Nanoscience in veterinary medicine.
Veterinary Research Communications, 31(Suppl. 1), 139–
144
S.S. Patil, K.B. Kore, Puneet Kumar (2009)
Nanotechnology and its applications in Veterinary and
Animal Science. Veterinary World, 2(12), 475-477
U.S. Food and Drug Administration (FDA) Report (2010)
Science and Research Special Topics

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 PRESS RELEASE
DAIRY SEMINAR – PUNE
A one day Seminar on “Challenges for Indian
Dairy Sector in the Coming Decade” was
organised by CLFMA of India on 24th January 2012
at Pune. Shri. Gopal Rao Mhaske, Chairman, Pune
Milk Co-operative Federation graced the occasion
as Chief Guest. Dr. C. S. Prasad, Vice Chancellor,
Maharashtra Animal & Fishery Science University,
Nagpur was Guest of Honour. After Inauguration and
the traditional lighting of Lamp, Mr. B. S. Yadav,
Chairman, CLFMA of India delivered the welcome
address. Dr. C. S. Prasad delivered the Keynote
address. Mementos were presented to the Chief
Guest and the Guest of Honour.
Technical Sessions were chaired by Dr. C. S.
Prasad and Co-chaired by Mr. Amit Saraogi. Dr.
Dinesh Bhosale presented an overview on activities
of CLFMA and set the tone for the presentations
which followed.
LIVESTOCK LINE, MARCH 2012
The first speaker Dr. S. Anandan, Senior Scientist,
National Institute of Animal Nutrition and Physiology,
spoke on Availability and Demand of Feeds and
Fodders in the country.
Mr. Vishvas Chitale, CEO, Chitale Dairy informed
the gathering about Challenges faced by the Private
Dairy Industry. Mr. Rahul Kumar, Managing Director,
Kaira Dist. Co-op. Milk Prod. Union Ltd. presented
the Challenges faced by Milk Cooperative Sector.
Mr. Girish Sohani President, BAIF, spoke on the
Challenges faced by NGO’s.
As a token of appreciation, memento were
presented to all speakers and sponsors of the
event. Mr. S.V. Bhave who was convener of the
seminar delivered Vote of Thanks.
The response to this successful seminar was
overwhelming with over 130 delegates in
attendance.
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