Microbiology World Issue 6

Microbiology World July – Aug 2014 ISSN 2350 - 8774

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Chief Editor

Mr. Sagar Aryal

(Founder) Ambassador, iversity

M.Sc. Medical Microbiology

St. Xavier’s College, Nepal

Editors

Mr. Saumyadip Sarkar

ELSEVIER Student Ambassador South Asia 2013 Ph.D Scholar (Human Genetics)

India

Mr. Avishekh Gautam

Ph.D Scholar Hallym University, South Korea

Mr. Manish Thapaliya

Ph.D Scholar China

Mr. Sunil Pandey

ELSEVIER Student Ambassador South Asia 2014 B.Sc. Medical Microbiology

Nobel Medical College, Nepal





Table of Content

Page No.

Ebola Hemorrhagic Fever 4-7

Chemically Programmed Antibodies 8-15

Interview with Dr. Leonard Mermel 16-19

Dye reducing activity of Microorganisms 20-22

Ebola: Most threating virus 23-24

Aromatherapy 25-32

Recent Diagnostic Techniques of

Subclinical Bovine Mastitis 33-38

Cells of Immune System 39-45





Ebola Hemorrhagic Fever

Jeny K. John1, Jobin Jose Kattoor1, Rekha V1, Aswathi P. B1, Aron Jacob2

1 PhD scholars, Indian Veterinary Research Institute, Izatnagar, Bareilly, U.P.

2 M.V.Sc Scholar, Indian Veterinary Research Institute, Izatnagar, Bareilly, U.P

Introduction

Ebola fever is a hemorrhagic fever affecting humans and non human primate species like

monkeys, gorillas and chimpanzees. Ebola hemorrhagic fever is a virus disease caused by virus

of family Filoviridae and genus Ebolavirus. The virus was first identified in 1976 in Democratic

Republic of the Congo near the Ebola River. Occurrence is sporadic in nature. Human disease

was confined in Africa. Disease is zoonotic in nature, bats are considered as the natural

reservoir host.

Causative agent

Ebola virions are linear, negative sense, single stranded RNA with inverse complementary 3'

and 5'termini (Pringle, 2005). Ebola virions are filamentous which appear in the shape of a

shepherd's crook or U or "6", and they may be coiled, toroid, or branched (Kiley et al., 1982).

There are 5 subtypes of Ebola virus: Zaire ebolavirus, Bundibugyo virus, Sudan virus, Taï

Forest virus and Reston virus. Among these, first four are known to cause disease in humans

and restricted in Africa and the fifth one was identified from cynomolgous monkeys from

Philippines.

Transmission

The primates get infection from bats, and humans will acquire infection from infected primates

through handling of diseased animals, through contact with infected body secretions, organs

and blood. Also aerosol route of transmission are reported in non human primates (Jaax et al.,

1995). Ebola-Reston virus mainly spread through aerosol route, which was first identified in a

research facility for primates in Virginia. Human to human transmission are possible through





contact with infected body fluids, organs and use of contaminated needles and syringes in

hospitals/clinics. Semen was also a source of transmission for around 2-3 months.

Outbreaks

Ebola Hemorrhagic fever (HF) has been reported from Democratic Republic of the Congo,

Gabon, Sudan, Ivory Coast, and Uganda, West African countries such as Nigeria, Guinea,

Sierra Leone and Liberia. Ebola – Reston first caused disease in monkeys in a research facility

in the United States and Italy in which monkeys were imported from the Philippines.

Major outbreaks

Year of outbreak Virus type Country

1976 Ebola-Zaire Zaire

1976 Ebola-Sudan Sudan


1994 Ebola-Zaire Gabon

1995 Ebola-Zaire Zaire

1996 Ebola-Zaire Gabon

2000-2001 Ebola-Sudan Uganda

2001 -2002 Ebola-Zaire Gabon, Republic of the Congo

2002-2003 Ebola-Zaire Republic of Congo


2007 Ebola-Zaire Democratic Republic of the Congo

Dec 2007 -Jan 2008 Ebola-Bundibugyo Uganda

2014 Ebola West Africa





Clinical signs

The incubation period for Ebola virus was around 2 days – 3 week. Case fatality rate was

around 50-90%. Sudden onset of illness and initial clinical signs includes fever, headache,

chills, joint and muscle aches, sore throat, arthritis and weakness, diarrhea, vomiting, and

stomach pain. Later stages hemorrhages from eye, ear, nose, gastrointestinal bleeding, genital

bleeding, and rashes over body surface. Also signs of coma, shock and disseminated

intravascular coagulation. Death occurs mainly due to shock.

Diagnosis

Due to non specific early clinical signs of Ebola hemorrhagic fever, diagnosis was difficult during

the early period. If a case was suspected, immediately isolate the patient and notify to health

authority. The samples suspected of Ebola hemorrhagic fever should be handled only in

biosafety level-4 laboratories. Initial tests include complete blood count, electrolytes, blood

clotting tests, liver function tests. Followed by virus isolation in cell culture, RT-PCR, serological

tests includes antigen-capture enzyme-linked immunosorbent assay (ELISA) testing, IgM

ELISA, indirect fluorescent antibody (IFA). Test should be conducted only in specialized

laboratories with biosafety level 4 facilities.

Treatment

There is no specific antiviral therapy available for Ebola virus, only supportive therapy is

available. Therapy for managing shock includes intravenous fluids and various medicines. Blood

transfusion is required if prolonged bleeding occurs. Balance the patient’s fluids and

electrolytes, maintaining their oxygen status and blood pressure. Treatment should be directed

towards the clinical signs. Shock, hemorrhage, neurological signs, high viremia and pregnancy

confer a poor prognosis.

Prevention

The prevention and control of Ebola HF faces many challenges, due to the lack of exact details

about the reservoir host. Present social and economic situation supports the spread of an

epidemic within health-care facilities. Therefore, health-care providers must be able to recognize





a case of Ebola HF as early as possible. They should be also facility for conducting diagnostic

tests and should adopt practical viral hemorrhagic fever isolation precautions or barrier nursing

techniques. These techniques include the wearing of protective clothing, such as masks, gloves,

gowns, and goggles; the use of infection-control measures, including complete equipment

sterilization and the isolation of Ebola HF patients from contact with unprotected persons. Ebola

virus can stay alive in liquid or dried material for a number of days and also in freezing or

refrigeration conditions. Ebola virus can be inactivated by UV radiation, gamma irradiation,

heating for 60 minutes at 60 °C or boiling for five minutes. The virus is susceptible to sodium

hypochlorite and disinfectants.

Reference

1. Jaax, N. et al. "Transmission of Ebola virus (Zaire strain) to uninfected control monkeys

in a biocontainment laboratory." The Lancet. 346: 1669-1671.

2. Pringle, C. R. (2005). "Order Mononegavirales". In Fauquet, C. M.; Mayo, M. A.;

Maniloff, J.; Desselberger, U.; Ball, L. A. Virus Taxonomy – Eighth Report of the

International Committee on Taxonomy of Viruses. San Diego, US: Elsevier/Academic

Press. pp. 609–614.

3. Kiley, M. P., Bowen, E. T., Eddy, G. A., Isaäcson, M., Johnson, K. M., McCormick, J. B.,

Murphy, F. A., Pattyn, S. R., Peters, D., Prozesky, O. W., Regnery, R. L., Simpson, D. I.,

Slenczka, W., Sureau, P., van der Groen, G., Webb, P. A., Wulff, H. (1982). "Filoviridae:

A taxonomic home for Marburg and Ebola viruses?".Intervirology. 18 (1–2): 24–32.





Chemically programmed antibodies: A next

generation antibody therapeutic

Vikas Gupta*a, Vinod Kumar Singha, Mukesh Bhatta , Vipin kumar Upadhayayb and

Utkarsh kumar tripathib

a-Division of virology, IVRI, Izatnagar, Bareilly, UP(243122)

b-Department of LPM, NDRI, Karnal, Haryana

*Corresponding Author: Phd scholar, Division of virology, I.V.R.I. Izatnagar Bareilly UP.

Email: [email protected]

1. Introduction:

World is facing different type of fatal and chronic disease since ancient time. To overcome these

diseases, demands for therapeutic agents are increasing day by day. A therapeutic agent to be

sufficient effective, it should have some important characteristic such as its site specific action,

sufficient serum half life, low dose with no adverse effect and most importantly economically

attractive. There are myriads of therapeutic agents, among them small therapeutic molecules

and monoclonal antibodies are in trend for the treating of disease. Small therapeutic molecule

have medium to high specificity to target, easily accessible to recessed site, unlimited diversity

and cheap but its serum half life is very less. In compression to small therapeutic molecule,

monoclonal antibodies have high target directed action, tuneable valency, effector function,

long circulatory half life but they have limited diversity , rarely reach to recessed site and costly.

So to get a good therapeutic agent, having long half life, unlimited diversities, target directed

action with high specificity, tuneable valency with effector function and economically attractive,

medicinal chemistry and protein engineering methods blended, yielding a new therapeutic agent

called as chemically programmed antibody. Chemically programmed antibodies are made up of

small therapeutic agents (synthetic agents) and antibodies. It has superior properties in

compression of individual component and target biding is mediated by synthetic agents, so

called chemically programmed antibodies.



mailto:[email protected]



2. Architecture of chemically programmed antibodies:

Chemically programmed antibodies are made up of synthetic agents, produced by medicinal

chemistry and antibodies, manufactured by protein engineering methods. So, they have major

two components, in form of antibody component and synthetic component.

2.1. Antibody component:

It includes monoclonal antibodies as whole or its fragment such as Fc or Fab. It acts as carrying

moiety for synthetic component. It provides circulatory half life, bivalency and effecter function of

conventional antibodies and reactive centre for the biding of synthetic component. Antibody

component is produce by reactive immunization and rational designing methods.

Reactive immunization is mean for the development of catalytic antibodies with the help of

reactive immunogens 1-3 diketone and venylenedine hapten with carrier are used as reactive

immunogen. The produce antibody has catalytic activity which mimics aldolase activity and

binding of synthetic component to the reactive centre of catalytic antibody component is

covalent and reversible in nature. There are mainly three types of reactive centre in antibody

component, lysine at paratope of antibody produce through reactive immunization and

selenocystein and cystein reactive centre at C or N terminal of antibody fragment produce

through phage display or native chemical ligation. These reactive centres provide site for the

binding of different synthetic component via reactive group. For conjugation of different

synthetic drugs to monoclonal antibody, require different specific monoclonal antibodies against

each type of target drug while by chemical programming methods, it can be avoided as different

synthetic component equipped with reactive group bind at reactive centre of antibody

component.

Fig: A Fig: B





Fig:1: Comprission between monoclonal antibodies and chemically programmed antibodies to

bind with different type of synthetic component. Fig:A depecting requirement of different types

of monoclonal antibodies for each type of synthetic component, Fig:B depecting same antibody

produce from reactive immunization can be used for different type of synthetic components.

2.2. Synthetic component:

It acts as binding moiety and has three components; pharmacophore, reactive centre and linker

group. Pharmacophore is the main binding moiety for the target site, it may be any peptides,

peptidomimmetics, small or large oligonucleotides and small therapeutic drugs having binding

specificity and affinity to extracellular (membrane bound or secreted) antigens. Reactive group

permits site specific and covalent binding at reactive residue of antibody component. Linker acts

as spacer for pharmacophore and reactive group.

3. Molecular assembly of chemically programmed antibodies:

There are mainly three types of molecular assembly of chemically programmed antibodies; IgG,

Fc and Fab based.

3.1. IgG based: In this type of molecular assembly pharmacophore bind at the paratope of

convention mAb via reactive lysine centre.

3.2. Fc based: It is most common type of assembly in which pharmacophore replaces the Fab

part and bind at C or N terminal with cysteine or selenocysteine reactive centre.

3.3. Fab based: It is a new type of molecular assembly pharmacophore bind with C terminal

selenocystein residue. Fab based chemically antibodies acts as carrier and targeting moiety

simultaneously and they are used as bi-specific chemically programmed antibody.

Fig:2: Different type of molecular assemblies of chemically programmed antibodies.





In bi-specific chemically programmed antibodies, one or both specificities are mediated by

synthetic component to target two epitopes or surface antigens. They have many type of

architecture based on antibody component. A first group among these utilizes two different

monospe-cific synthetic components. These are either conjugated to two identical unique

reactivity centers or to two orthogonal unique reactivity centers. A second group of bispecific

cpAbs utilizes a bispecific synthetic component. Published examples include trifunctional

synthetic components that combine specificities for two different extracellular antigens with a

reactive group. A third group of Fab bispecific cpAbs makes use of antibody components with a

paratope that remains untouched by chemical programming. Conjugating a monospecific

synthetic component to a unique reactivity center in these antibody components affords two

antigen-binding sites; one provided by the antibody component and one provided by the

synthetic component Fab based bispecific abtibodies brings target and effector cells into close

contact to for cytolytic synapse. They may be monovalent bi-specific or divalent bi-specific.

Fig:3: Different form of chemically programmed bispecific antibodies.





4. Targeting agents for chemically programmed antibodies:

Targeting molecule may be membrane bound antigens, integrin molecules and different

miscellaneous molecule. Membrane bound antigen includes gp120 of HIV neuraminidase

glycoproteins of influenza virus chemokine receptors (CCR5) besides these opiodes receptors

and luteinizing hormone receptor can be used. Integrin molecule include several integrins,

which are expressed on tumor cells and tumor endothelial cells, have high-affinity binding sites

for tripeptide motifs, such as RGD (integrin αvβ3, αvβ5, αvβ6, and α5β1) and LDV (integrins

α4β1 and α4β7). These tripeptide motifs are present in extracellular matrix and cell surface

proteins that bind to integrins. As linear or cyclic peptides, they potently antagonize these

interactions and induce apoptosis. Miscellaneous agents includes angiopoietin-2, vascular

endothelial growth factor (VEGF), and placental growth factor-1

5. Characteristic of chemically programmed antibodies:

Antigen recognition by synthetic component

Unlimited chemical diversity

Long circulatory half life

Medium manufacturing cost

Have effector functions

High target binding specificity and affinity

6. Applications of chemically programmed antibodies:

6.1. As a long lasting and potent inhibitors of influenza neuraminidase:

Influenza viruses have two glycoprotein surface spikes, hemagglutinin and neuraminidase.

Hemagglitinin is responsible for bind and fusion of virus on cell surface with help of sialic acid

receptor of cells while neuraminidase is responsible for the release of denova virion from

infected cell and its further spread to other cells. For the control of influenza infection

vaccination plays primary role but due mutations and seasonal incidence, it have some

limitations as vaccine must be read before the influenza season and it is not able to cope up the

new variants of influenza. So along with vaccination, anti influenza therapeutic agents are also





required. Currently, small molecule drugs aim to inhibit neuraminidase are used as therapeutic

agents as neuraminidase catalytic site is resistance to mutation for maintaining the enzymatic

activity. Neuraminidase inhibitors such as oseltamivir and zanamivir are frequently used as anti-

influenza drugs but they have very short life, so frequent doses are required. To avoid frequent

dosing and enhancement of short of serum half life of drug, β-lactam functionalized

neuraminidase inhibitors are conjugated to catalytic aldose monoclonal antibodies 38C2. This

conjugated chemically programmed antibodies bind with catalytic site of neuraminidase and

inhibit the release of newly formed viruses.

Fig:4: Chemically programmed antibody conjugated with Zanamivir antiviral drug.

6.2. As a enhancer of bread and potency neutralization of anti-HIV-1 antibodies and CD4

IgG:

Human immunodeficiency virus-1 is major threat to human being as more than 2 million people

die every year, and more than 33 million individuals are infected worldwide. Highly active

antiretroviral therapy (HAART) is used for therapy of HIV-1but it have so many adverse effect

also. So, development of a potent and broadly acting biological agent as therapeutics might be

a solution. Broadly neutralizing monoclonal antibodies (BNmAbs) that recognize features

conserved across clades of HIV are promising starting points for the development of

immunotherapeutic agents against HIV-1. But their neutralizing capacity is upto 70 to 80%. If a

BNmAb could be modified to inhibit HIV in some different ways, then they can be used as good

therapeutics. HIV-1 entry in side CD4+T cells through receptor mediated mechanism with help





of CD4 receptor along with CCR5 and CXCR4. A promising additional blockade to HIV-1

infection that should complement the targeting of viral proteins is the targeting of host proteins

required for viral entry and replication. A number of small-molecule inhibitors of the HIV-1

coreceptors CCR5 and CXCR4 have been developed and one CCR5-targeting drug has been

approved. CCR5-targeting small molecule, such as meraviroc or aplaviroc is conjugated to

BNmAbs and CD4-IgG, which function as bispecific chemically programmed antibodies. These

bispecific chemically programmed antibodies bind to CCR5 co-receptor and inhibit the entry of

virus in side T cells along with neutralization of HIV-1.

Fig:5: Mechanism of action of chemically programmed antibody conjugated with CCR5

antagonists

6.3. Chemically programmed antibodies as recruiter and activator of T cells:

Biscepicific monoclonal antibodies against CD3 of T cell are conjugated with some receptors

analogues of intergin α4β1 or folate-1 receptor (FOLR1) of malignant tumor cells. Bispecific

CD3 monoclonal antibodies are conjugated with peptidomimetic LLAP2, analogue of integrin

α4β1 binding residue of extracellular matrix bind with integrin α4β1 on tumor cell surface and

recruit the T cells at vicinity of tumor cell. Due to binding of bispecific chemically programmed

antibodies on tumor cell, along with T cells, there is formation of cytolytic synapsis, which

further activate T cell and ultimately lead to cytolysis of malignant tumor cell.





6.4. Chemically programmed antibodies as anti-cancer therapeutics:

Different types of intregin expressed on the cell are responsible for the adhesion, proliferation,

migration differentiation and cell death. Intergin mediated cell-cell binding and cell matrix

attachment with their cognate ligands and expression of these receptors are normal and well

control in normal cells. In contrasts to it, in malignant cells among these intergins, some are

over expressed such as αvβ3 andαvβ5 which are responsible for angiogenesis and matastasis

of maliganant cells. So, these over expressed intergins are the promising target for the

anticancer therapeutics. Anticancer drugs have very short half life and require in high dose, due

high dose they causes cytotoxicity to normal cells also. To avoid this, peptidomimetic drugs

analogue, SCS873 of RGD tri-motif which bind with αvβ3 and αvβ5 intergin, conjugated to the

catalytic aldolase mab 38C2.These conjugated antibodies binds with αvβ3 and αvβ5 intergin

present on tumor cells and Interfere interaction between integrin and extracellular matrix

proteins which leads to Apoptosis of tumor cells and inhibition of angiogenesis.

7. Conclusions:

They are versatile in nature, provide instant immunity, same mAb can be used for different type

of chemical programming. It can be used against two or more antigen at a time via bispecific

chemically programmed antibodies. In last they are economical and less or no toxic.





Interview with Dr. Leonard Mermel

Q) Dr. Leonard Mermel is a well known professor, doctor and researcher

at Brown University. You are well known from the background of

nosocomial infection research. Before knowing more about your research

experiences would like to start off with your early childhood days. How you

used to take medical science during your schooling? How your parents

used to influence you about your higher studies and then going for

research?

Comment: My first interest in becoming a doctor occurred when I was around 10 years of age

seeing my grandmother in a hospital after having a myocardial infarction. I was very close to

her and felt helpless and I told her and my mother then that I want to be a doctor when I grow

up. My family always stressed education, particularly my father who is a told us repeatedly that

receiving a higher education was of the utmost importance. I had great teachers along the way

who served as role models. My interest in clinical and translational research developed during

my infectious diseases fellowship under the tutelage of Dr. Dennis Maki.

Q) You have received multiple awards like Young Investigator award from Society for

Healthcare Epidemiology of America, Teaching awards from The Warren Alpert Medical School

of Brown University and Mentor Scholar award from Society for Healthcare Epidemiology of

America. If you could recall, does these awards gave you zeal to work forth with your research?

Comment: Most certainly, it is an honor and privilege to engage young, bright minds and

mentoring them to have the immense satisfaction in carrying out a successful clinical research

project. There is no greater pleasure as a professor and I feel a strong commitment to such

endeavors.





Q) The techniques used earlier had been more complicated. With the invention of sequencing

techniques, PCR and multiple analysis technique helped people to overcome hurdles of

research nowadays. How you compare the earlier techniques and the current techniques of

research you use.

Comment: Techniques in research should be applied to the question at hand. Knowing which

ones to used reflects collaboration with mentors, mentees and colleagues.

Q) Looking back one of your research published in Infection Control and Hospital Epidemiology

(2010) on identified Methicillin-Resistant Staphylococcus aureus (MRSA) in patients HIV-

infected and hemodialysis patients. In that research findings one in every three individuals have

the prevalence of MRSA in different populations. MRSA ranks 1 in spreading hospital acquired

infection. Does Hospitals can overcome this problem of defeating MRSA with a proper drug?

Comment: Reducing risk of MRSA colonization and infection in hospitalized patients depends

on a strong infection prevention and control program. We have an aggressive program for

MRSA and we have tremendously reduced risk of such infections to our patients over the last 2

decades.

Q) You had been visiting professor at University of Wisconsin Hospital and Clinics, Madison and

Baystate Medical Center, Springfield,MA. How you interact with your students about the

research you carry out? Do you influence your students to think over any complications of

medical research?

Comment: I emphasize that finding a project that is within the scope of the resources and time

frame are critically important as well as finding a mentor who has the time and expertise to

assist in that endeavor.

Q) During the course of your research experience have you ever come up with a bacterium

which you find very hard to disinfect it because of its high resistance (apart from MRSA)?





Resistance of bacterium is acquired based on the resistance genes present in their plasmid.

Have you come up with any research technique to reduce copy numbers of the plasmid of those

bacteria?

Comment: My research has not focused on reducing plasmid transmission of resistance genes.

However, I have collaborated on research looking at novel anti-infective compounds such as

using catheters coated with 5-fluorocytosine as well as more recently looking at novel

compounds with antimicrobial properties to use as antimicrobial lock solutions to prevent or treat

catheter infections.

Q) Highlight one more important and spectacular research of yours, ‘Infection Prevention and

Control during Prolonged Human Space Travel’ published in Clinical Infectious Diseases (2012).

Prolonged space flight and microgravity can provide unique advantages to germs specially

Salmonella or Pneumococcus outbreak. We would like to know how encouraging was that

journey in solving the entire mystery behind microbial advantage in space.

Comment: It is my great fortune to work with bright individuals at Johnson Space Center and

discuss prevention of infections during prolonged human space flight. This is a most interesting

issue as microgravity of space travel presents tremendous challenges and in some cases novel

solutions to mitigate such risk.

Q) Being a professor, researcher and doctor you might have faced varied controversies during

the course of your research. How you used to overcome them and stayed focused in your work?

This will definitely provide a better understanding and will grow motivation among young

researchers and students.

Comment: Controversy abounds in science. It is important to try to remain objective and open-

minded to other points of view, even if in conflict with one’s own research findings. History will

ultimately reveal what was the right direction, but at the time, it may be hard to know which is

why replication of scientific findings by other investigators is essential in the scientific endeavor.





Q) While concluding your journey of research, we would like to know your personal message

towards young researchers and students of medical microbiology.

Comment: I hope I am not concluding my journey but continuing down the same rewarding

path as I have done over the last quarter century. My message is find a good mentor, be

patient, humble, forward-thinking, keep up with the medical literature and acknowledge those

who help you along the way.

Q) There is obvious a wonderful happy life behind research which help in focusing any work.

Thereby would like to know about your life apart from research.

Comments: I have many other interests, I enjoy biking, hiking, surfing, photography, music, and

I have a moderate-sized vegetable garden and numerous fruit trees all of which I planted,

manage, and harvest.

Interview Taken By:

Saumyadip Sarkar

Science Communicator and Reviewer,

Microbiology World,

www.microbiologyworld.com

[email protected]



mailto:[email protected]



Dye reducing activity of Microorganisms

Mr. Harshwardhan M. Shrungare

Technical assistant,

Microbiology Research lab,

R. A. College, Washim

Introduction:

It is prove that water is life but now a day that life is threatening due to the advancement in

industrialization. The increase in demand of chemical dyes, fertilizers also increases the

manufacturing and its supply by many industries. During this manufacturing gallons of dyes and

chemicals are incorporated into the river that can cause damage to the environment. Pollutants

mainly include acids, bases, toxic organic and inorganic dissolved solids and colors and their

sources are varying. Textile industry is one of them which extensively use synthetic chemicals

for dye production. Fortunately microbes have ability to reduce these dyes and hence an

attempt is made in Microbiology Research Lab, R. A. College, Washim as a part of project.

Consequences of dyes and chemical in rivers:

Textile industries generate high volume of waste water and dyes. About 10,000 different dyes

and pigments produced annually and about 10% are lost in river via waste water of industry.

The strong color of this waste water is most serious problem because it is the most undesirable

character of water. The disposal of such waste water containing dyes into the river causes

damage to the environment. Generally dye can be describe as, a colored compound that has an

affinity to the substrate to which it is being applied. Dyes affect the photosynthetic activity in

aquatic habitat; it is toxic to aquatic life and causes dangerous effect on all living system. Some

physicochemical methods are available for water treatment including ozonation, photo-

oxidation, adsorption, activated carbon, froth flotation etc. but these techniques creates

secondary disposal problem and are expensive.





Significance of bioremediation of dyes:

Considering above drawbacks, the concept of bioremediation of textile effluent has gain much

attention. The process of microbial de-colorization is eco-friendly, cost competitive and effective

method for effluent treatment. Hence an experiment was carried out to check the efficiency of

the process.

Experimental procedure and results:

In the performed experiment, bacteria such as Pseudomonas and Bacillus have been used to

degrade azo dyes from industrial effluent. Micro-organisms from rhizospheric soil is grown on

nutrient agar modified with effluent and after incubation microbes which decolorizes or grown on

medium are collected and proceed for further application. Two types of cultures were prepared

from the growth obtained, one is free culture and other one is immobilized cell culture. Both

cultures were mixed with industrial effluent and after 6-7 days it was found that the effluent is

decolorizes. The maximum dye de-colorization ability of free and immobilized cells were

analyzed and it was found that immobilized cell culture have maximum activity. Further, cultural

and morphological analysis was also done and the cultures were confirmed as Bacillus,

Clostridium and Pseudomonas for textile effluent de-colorization.

References:

1. Caldwell, B., and M. P. Bryant. 1966. Medium without rumen fluid for nonselective

enumeration and isolation of rumen bacteria. Appl. Microbiol. 14:794-801.

2. Cerniglia, C. E., J. P. Freeman, W. Franklin, and L. D. Pack. 1982. Metabolism of azo

dyes derived from benzidine, 3,3'- dimethylbenzidine and 3,3'-dimethoxybenzidine to

potentially carcinogenic aromatic amines by intestinal bacteria. Carcinogenesis 3:1255-

1260.





3. G. McMullan, C. Meehan, A. Conneely, N. Kirby, T. Robinson, P. Nigam, I. Banat, R.

Marchant, W. Smyth 2000. Microbial decolourisation and degradation of textile dyes.

Applied Microbiology and Biotechnology July 2001, Volume 56, Issue 1-2, pp 81-87.

4. King-Thom Chung* and S. Edward Stevens Jr. 1993. Degradation azo dyes by

environmental microorganisms and helminthes. Environmental Toxicology and

Chemistry Volume 12, Issue 11, pages 2121–2132,

5. Tim Robinson, Geoff McMullan, Roger Marchant, Poonam Nigam 2000. Remediation of

dyes in textile effluent: a critical review on current treatment technologies with a

proposed alternative. Bioresource Technology Volume 77, Issue 3, May 2001, Pages

247–255.





Ebola: most threating virus

Anu Shree Varshney

Now a day everyone was hearing about Ebola virus which was spread all over Sierra Leone,

Liberia, Guinea and Nigeria. You know how this virus is spread, are you safe from the infection

of this virus? If you don’t know about this then what are the symptoms of this virus? How can

you protect yourself not getting infected with this virus? The biggest question is your country

your area is safe from Ebola virus? And most important whether you exactly know what was it?

When someone talk about a Ebola virus people take it lightly by saying “It is not prevailing in our

country , so there is no point of discussing it ” There is no awareness program for it i.e. what it is

, what are the symptoms , if you got infected what you should do first ?

Ebola is also called as Ebola haemorrhagic

fever. Ebola first appeared in 1976 in 2

simultaneous outbreaks, in Nzara, Sudan, and

in Yambuku,. The latter was in a village situated

near the Ebola River, from which the disease

takes its name. Genus Ebolavirus is 1 of 3

members of the Filoviridae family (filovirus),

along with genus Marburgvirus and genus Cuevavirus. Genus Ebolavirus comprises 5 distinct

species.Ebola is a spread from infected person to healthy person. Ebola enter into the human

population through the animal which are at higher risk i.e. monkey, fruits bat, chimpanzee,

gorillas. So it is advisable not to handle the infected animal as the secretion of these animal i.e.

urine, saliva and when a person come in contact with the blood of these animal get infected with

the Ebola virus. When the secretion of the infected person come in contact with the healthy

person it transfer the virus in the healthy person this is how this virus spread. Health workers

are at higher risk of getting this infection if they are not protection equipment like gloves etc

when treating with Ebola infected patient. Burial ceremonies in which mourners have direct

contact with the body of the deceased person can also play a role in the transmission of Ebola.





Now after knowing what is

Ebola virus and how one can

get infected with these virus

.One should know what the

symptoms of this virus are?

Early signs and symptoms

include: Fever, Severe

headache, Joint and muscle

aches, Chills, Weakness. Over

time, symptoms become

increasingly severe and may

include Nausea and vomiting,

Diarrhea (may be bloody),

Red eyes, Raised rash, Chest

pain and cough, Stomach

pain, Severe weight loss,

Bleeding, usually from the

eyes, and bruising (people

near death may bleed from

other orifices, such as ears, nose and rectum), Internal bleeding. There is no vaccine for Ebola

virus, many vaccines had been tried but these were not effective in providing any relief from this

virus. The Only way to protect is to raise awareness about it. During this outbreak, most of the

disease has spread through human-to-human transmission. Several steps can be taken to help

in preventing infection and limiting or stopping transmission: Listen and follow the directive

issued by the minister of health. If you choose to care for an ill person in your home, notify

public health officials of your intentions so they can train you and provide appropriate gloves

and personal protective equipment (PPE), as well as instructions as a reminder on how to

properly care for the patient, protect yourself and your family, and properly dispose of the PPE

after the use. So overall personal safety , hygiene at personal level is very important in

protecting from not getting infected with the Ebola virus.





Aromatherapy

*Hasnain Nangyal1, Upvan Bhushan2, Ammara Nawaz3

1Department of Botany, Hazara University, Mansehra

2Department of Botany, University of Jammu, India

3Department of Zoology, Punjab University, Lahore

Aromatherapy

Aromatherapy is composed of two Greek letters, Aroma (fragrance) and therapy (treatment).It

means that treatment with different fragrances.

Does aromatherapy really work?

It is use of aromatic compounds like

essential oils that derived from plant

sources that help to promote the

physiological and physical well-being. It is

used as a source of alternative medicine

in form of concentrated essential oils.

These essential oils are extracted from

different parts of plants like flower,

leaves, seed and twigs etc. Essential oils does not means that it have some nutritional type

values, but it means that they are extracted from plants that have aromatic and volatile

components that may contain antibiotics, vitamins, to some extant hormones and antiseptics.

Fragrances of these essential oils have been used to reduce the mental stress and help to

relieve the tension since many centuries. Further research and experimentation, now it is

proved that these essential oils have been play its role in reduce the dandruff and enhance the

healing properties. Most commonly used essential oils are lavender, rose, orange and sand

wood oils. So aromatherapy massage helps to reduce the anxiety and depression





History of Aromatherapy

History of aromatherapy is as old as history of

human being. History of aromatherapy is

started from circa (460-377 BC) era that first

physician of Egypt and had belief that illness

was caused by supernatural forces.So for the

treatment of such supernatural forces different therapies recommended, and aromatherapy is

one of them. More than 200 herbs had studied by Hippocrates that used for source of essential

oils in aromatherapy.

Over 2000 years later such therapies was employed in Egypt in form of bath, massage and

even embalming bodies. Between 18th and the 25th Dynasty (1539-657 BC), the Egyptians

continued to refine the use of aromatics in incense, medicine, cosmetics, and finally perfumes.

Similarly in 1928 this therapy is introduced in France by a chemist, René Maurice Gattefossé. In

France this therapy got importance when Rene become interested in healing proprieties of

essential oils that extracted from different plant sources. He uses the technique of distillation

with the help of which, he extract the aromatic compounds in form of essential oils from the

plants that have wonderful smell. Then a question arouses in His mind how the smell of these

aromatic compounds helps in relaxation of body and mind. To find out the question of this

answer, accidently his hand burnt during the laboratory work then he used the lavender oil for

relieving the pain it was amazing for him within few minutes sensation of burning was gone.

With the passage of time and advancement in science and technology, Aromatherapy get more

importance in 20th century and more working will be continued on aromatherapy.

Why we use Aromatherapy

Basic purpose of aromatherapy is to keep the mind and body in good state. It helps to promote

the sensory experiences of massage and relieve the mental tension. The fragrance of aromatic

compounds helps to maintain the outlook of limbic system that helps to regulate the mental





stress. Aromatic compounds that obtain from the different plant sources help to regulate the

emotional condition and mental stress where the synthetic medicines currently fail.

Essential oils

These are the extracted material obtain

from the different parts of plants by

different methods. Basic component of

these essential oils are aromatic

compounds and its derivates that have

healing properties. Essential oils are

volatile in nature that evaporated at room temperature that left a specific fragrance. Essential

oils are differing in chemical compositions that are found in oils of fat and fatty acids.

Components of Essential oils

There are following the important components that found in Essential oils.

Terpenes, Phenols, Ketones, Ether, Ester, Aldehyde, Alcohols

Terpenes: There are two types of terpenes; 1) Monoterpenes & 2) Sesquiterpenes

Monoterpenes: These compounds are found all most in all essential oils and have 10 carbon

atom structures with at least one double bond. In the presence of air such compounds are

readily oxidized.

Sesquiterpenes: Such compounds have almost 15 carbon atom structures that are more

complex. It is found in German Chamomile.

Phenols: Mostly the derivatives of phenols are found in essential oils such as thymol, eugenol

and carvacrol. Such oils have antiseptic and disinfectant properties.

Ketones: Such compounds are found in hyssop, eucalyptus and rosemary oils. The essential

oils that have such ketonic compounds must be avoided to use during pregnancy.





Ethers: Such compounds are found in tarragon and basil oils.

Esters: The essential oils that have such ester compounds have fruity smell and have sedative,

antibacterial and antifungal properties.

Aldehyde: Such compounds are found in citrus fruits and lemon oils. Such essential oils have

anti-fungal, anti-inflammatory, disinfectant, sedative yet uplifting therapeutic qualities.

Alcohols: It has two types; monoalcohols and Sesquialcohols

Monoalcohols: Such compounds are found in juniper oil and tea tree oils

Sesquialcohols: Such compounds are rarely found in essential oils such as chamomile oils

Extraction of Essential oils: Following the techniques is used for extraction of essential oils

from plants.

Distillation: Plant material is soaked and after that process of distillation is done. The process

of distillation may be steam distillation and hydro distillation. In case of steam distillation steam

is passed through condenser then liquefies the oils. While in case of hydro distillation plant

materials is soaked and make a broth. But this method is not suitable because material is

overcooked. This method is suitable for extraction of oils from seeds or stem.

Solvent extraction: Different organic solvents are used for extraction of oils like Linden

Blossom and Jasmine

Resinoids: these are prepared from the dead organic plants that basically have hydrocarbons

play its role for extraction of essential oil from plant source.

Mode of action of Aromatherapy

Total of the 15% of air that we inhaled is enter in nose where olfactory receptors transport

odors’ to apart of the brain called the limbic system that connected to our mood and emotion. In

case of aromatherapy essential oils have specific odor due to chemicals that unlock the

emotions .These small molecules are absorbed in blood through lungs where it diffuse to





tissues .The aroma from the oils sends a message to limbic system which is center of

controlling the emotions, memory and sexual arousal.

Fight and Flight hormones involve in Aromatherapy

Epinephrine and nor epinephrine hormones are called normally fight and flight hormones that

play a crucial role for survival of life. During the emotional and physiological stress, over

production of these hormones weaken the immune system by reducing the T helper cell and

inhibit the natural killer cells. Now a day's research on aromatherapy may leading to this aspect

the persons who treated by aromatherapy boost up their immune system during the depression

and stress conditions. Regular treatment of aromatherapy and massage with essential oils helps

to break the depression cycle and boost up the immune system.

Applications of Aromatheorapy

Several different methods are adopted to use the essential oils in aromatherapy.

Full-body baths: 5 to 10 drops of essential oils are mixed in tub of water either hot or cold.

Hand or foot baths: 2 to 3 drops of essential oils are mixed in tub of water either hot or cold

and then soaked hand or feet in this water for 10 to 20 minutes.





Inhalations: This is apply in treatment of nasal problems such as sinus or allergic reactions.

Small amount of water boil and then add 2 to 3 drops of essential oils and inhale its steam.

Diffusion: In this technique special type of nebulizer used that help to disperse small droplets of

essential oil into the air. This is recommended to break down the depression cycle.

Massage: In this technique essential oils mixed with other carrier oils e.g wheat germ, avocado,

olive, safflower, grape seed, or Soya bean oil. A ratio that is commonly recommended is 2.5–5%

essential oil to 95–97.5% carrier oil.

Essential oils that commonly used: some of important essential oils presented there that mostly

commonly used

Roman chamomile oil;

It is helpful in treatment of skin diseases, menstrual pain and depression.

Peppermint oil;

It helps to relax the stomach muscles and gastrointestinal tract. Its acts as

an anti-inflammatory, antiseptic, and antimicrobial that make effective in

treatment of cold and flu symptoms.

Rosemary oil;

It helps in treatment of muscular, as well as low blood pressure,

gastrointestinal problems and headaches.

Lemon oil;

It acts as anti-stress and anti-depressant.





Eucalyptus oil;

It helps to aid respiratory system ailments by enhancing deep

breathing

Lavender oil;

Aroma therapists use it to treat respiratory problems, abdominal cramps,

depression, insomnia, tension-related problems, burns, sun-damaged skin,

and various types of skin infections.

Rose oil;

Good for headache, nervous tension, stress related conditions, insomnia,

and nausea. Use against broken capillaries, dry skin, and poor circulation.

Jasmine oil;

Relaxing and intoxicating. Jasmine is valued in skincare in aiding dry,

sensitive and irritated skin

Side Effects of Aromatheorapy

We should never take the essential oils orally as recommended by professional aroma

therapeutics. Aromatherapy can induce side effects, such as rash, headache, liver and nerve

damage, as well as harm to the fetus. Oils that have high phenolic contents such as cinnamon

can cause skin irritation. Dilute oil with water or a base massages oil (such as almond or





sesame oil) before applying to your skin, and avoid using near your eyes. In addition, essential

oils are highly volatile and flame able so they should never be used near an open flame.

Precautions

Some essential oils directly apply to the skin in form of perfume. Oils of orange and peppermint

cause irritation to the skin if applied in concentrated form. During the massage essential oils are

mixed with carrier or vegetable oils. A final precaution is to avoid taking essential oils internally

without a consultation with a physician or naturopathist. Citrus-based essential oils, including

bitter and sweet orange, lime, lemon, grapefruit, and tangerine, are phototoxic, and exposure to

direct sunlight should be avoided for at least four hours after their application. Before using

essential oils on the skin, individuals should perform a skin patch test by applying a small

amount of the diluted oil behind the wrist and covering it with a bandage or cloth for up to 12

hours.





Recent Diagnostic Techniques of Subclinical

Bovine Mastitis

Aron Jacob, Tshering Dolma, Rekha V, Aswathy P B and Jeny K John

Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh.

Introduction

Mastitis remains the most common and costly disease of dairy cattle (Oviedo- Boyso et al.,

2007). Treatment and control of mastitis is difficult due to the multiple causative agents i.e.,

more than 200 different organisms have been recorded in scientific literatures as being causes

of bovine mastitis. Mastitis results when pathogenic bacteria are able to gain entrance to the

udder, overcome the cows’ immune defences, establish an infection and produce inflammation

of udder secretory tissue. Contagious pathogens like Streptococcus agalactiae and

Staphylococcus aureus reside primarily in the udder of infected cows. Transmission is limited

only at the time of milking process. Environmental pathogens are common inhabitants of the

cow’s environment. The transmission can occur at any time including milking time, between

milkings, during dry period and prior to first calving in heifers.

Recent diagnostic techniques for detection of subclinical mastitis

Subclinical mastitis is difficult to detect due to the absence of any visible indications and

requires the availability of a rapid screening test for early disease detection (Viguier et al.,

2009).

Somatic Cell Count

The test measures increase in somatic cells in milk. SCC at quarter level gives better detection

performance than measuring SCC at cow level (Mollenhorst, H., 2010). Somatic cell counters

operate on the principle of optical fluorescence where ethidium bromide or propidium iodide

used stain nuclear DNA and the fluorescent signal generated is used to estimate the SCC in

milk. Esterase-catalyzed enzymatic reaction assay is also useful to determine the SCC.





Electrical Conductivity

This test measures the increase in conductance in milk caused by the elevation in levels of ions

such as sodium, potassium, calcium, magnesium and chloride during inflammation. Janzekovic

et al., (2009) indicated that the conductivity in individual quarters was <5.5 mS/cm for healthy

udders and >6.5 mS/cm for subclinical udders.

NAGase test

According to Pyrola et al., (2003) estimation of endogenous enzyme N- acetyl-β-D-

glucosaminidase is the most accurate of the indirect tests. It is an intracellular lysosomal

enzyme from neutrophils and epithelial cells. Production month, breed, lactation stage and milk

yield significantly affects NAGase activity. Compared to primiparous animals activity is higher for

multiparous animals. NAGase activity is higher at beginning of lactation and in late lactation.

Colorimetric and fluorometric assays have been developed to measure the elevated

concentration levels of these enzymes in milk (Larsen, 2005).Chagunda et al., 2005 states that

in mastitis NAGase tend to increase before the day of diagnosis and drop after treatment. The

enzyme activity tends to increase about eight days before diagnosis and more rapidly than

somatic cell count. So NAGase activity could be effectively utilized for diagnosis of mastitis.

Normal animals: 43.47nmol/ml mastitic: 69.42nmol/ml.

LDH estimation

Lactose dehydrogenase (endogenous) activity tends to increase 8 days before diagnosis and

more rapidly compared to somatic cell count. Compared to NAGase and SCC, LDH activity has

highest change in mastitis; SCC has the lowest (Chagunda et al., 2005). In healthy animals LDH

activity in milk is 485.94±13.66 IU/l, where as subclinical mastitis cases it is 1524.04±111.74

IU/l. The activity of LDH, NAGase and SCC could be used as early indicators of mastitis.

Production month, lactation stage and milk yield significantly affects LDH activity. Age at first

calving and parity didn’t have significant effect on enzyme activity. Compared to primiparous

animals’ activity is higher for multiparous animals. LDH activity is higher at beginning of lactation

and in late lactation than in mid lactation. Mohammadian (2011), states that the mean activity of

(LDH) was higher in milk from mastitic udders than in milk from healthy udders.





Spectrophotometry, colorimetric LDH quantification assay, ELISA are the tests used for

estimating LDH activity in milk.

L (+) lactate estimation

Lactate concentration may increase in a series of secretory, post secretory, physiological and

pathological reasons, still it can be used as a supportive parameter for diagnosis of subclinical

mastitis, and special attention must be given to physiological factors. Davis, et al., (2004)

described a rapid increase in lactate concentration during mastitis. Lactate can be used as

supportive parameter for mastitis detection (Grabowski et al., 2005). Milk sample should be

fixed properly in trichloroacetic acid to avoid post secretory lactate increase. Lactate is oxidized

to NAD+ and pyruvate and NADPH produced is measured photometrically. L+ Lactate content

in milk healthy: 0•1mM, subclinical: 0•7 to 1•5 mM, clinical: 3•3 mM. Bacteria also produce

lactate but it is D type rather than L type. Lactate together with pyruvate is used to estimate

bacterial count in milk.

Lactose estimation

In mastitis lactose content reduces to 2.5-2.83 %. According to Solverod (2005) a somatic cell

counts of more than 400000/ml corresponds with reduction in lactose content in same fraction

possibly because of damage of epithelium. Lactose content in milk can be used as indicator of

subclinical mastitis (Sharif et al., 2007).

Acute phase proteins - Milk Amyloid A

Lahtolainen, T., (2004) found that in experimental udder infections had a promising potential for

production of milk amyloid A as an indicator. A local synthesis of Milk amyloid A, a milk specific

acute phase protein in inflamed udder has been reported (Jacobsen et al., 2005). Petersen, et

al, 2005 stated that even though there is no overall difference in diagnostic performance the

somatic cell count and milk amyloid A have different diagnostic potential that could be depend

on factors such as nature of causative agents, milk kinetics during inflammation and degree of

tissue damage. M-SAA concentrations for healthy cows and cows with clinical mastitis were





reported also by Haghkhah et al., (2009) 9.90 and 105.12 μg/ml, respectively. MAA as a

potential physiological marker of subclinical mastitis (Gerandi, et al., 2009)

Diagnosis of mastitis by benzoic acid and sodium carbonate

Ayaz et al., (2005) described a method to diagnose mastitis using benzene sulfonic acid and

sodium carbonate solution as somatic cell count increase in mastitis sodium carbonate liquefy

DNA of these cells and release in solution to form jelly like substance with benzene sulfonic

acid. This test could be the cheapest and easy test to diagnose subclinical mastitis.

Cytokines analysis

TNFα, IL-1β, IL-6, IL-8 have been found to be released in bacterial mastitis For cytokine

analysis milk should be centrifuged at 2500 rpm for 20 min at 4°C to get fat free cell free milk

extract and should be kept at -30° C until assayed using ELISA or flurescencoptical method. In

a study conducted by Winter et al., 2005, found that during experimental challenge with

Staphylococcus epidermidis to ovine udders showed elevation of IL-8 in infected gland at 2hr

and peak at 8hr – 24 hr and will remain for 10wks and IL-1β transiently elevated at 1 and 2 day.

Conclusion

The challenge in treatment and control of mastitis is mainly due to presence of multiple

etiological agents and development of resistance to antimicrobial agent. Timely diagnosis and

prompt treatment can reduce the losses to the livestock economy by this disease syndrome.

Since the disease is of public health significance diagnosis of mastitis at the earliest is a

requisite for human health too.

References

1. Ayaz , M. M. and Akhtar, M (2005) Diagnosis of mastitis by benzoic acid and sodium

carbonate on pregnant and lactating mastitic animals. Mastitis in Dairy production

Current knowledge and future solutions.





2. Chagunda, M. G. G., Larsen, T., Bjerring, M. and Ingvartsen K. L. 2005 Changes in

lactate dehydrogenase, N- acetyl β D- glucosaminidase and somatic cell count in

relation to mastitis development in dairy cows. Mastitis in Dairy production Current

knowledge and future solutions.

3. Davis, S.R., Farr. V.C., Prosser C.G., Nicholas, G.D., Turner S.A., Lee, J. and Hart A.L.

2004. Milk L-lactate concentration is increased during mastitis. J.Dairy Res.78, p.175-

181.

4. Gerandi, G., Bernadini, D., Elica, A.C., Ferrari, V., Iob, L. and Segato, S. 2009. Use of

serum amyloid A and milk amyloid A in diagnosis of subclinical mastitis in dairy cows. J.

Dairy Res 76(4):p 411-17

5. Grabowski, N. Th., Redetzky, R.,Sulzer A., Hamann, J. and Kliein, G. 2005. Using milk

L(+) lactate as a diagnostic tool to detect mastitis in early lactation. Mastitis in Dairy

production Current knowledge and future solutions.

6. Haghkhah, M., Nafizi, S. and Jahromi, A. G. 2010. Evaluation of milk haptoglobin and

amyloid A in high producing dairy cattle with clinical and subclinical mastitis in Shiraz. J.

Comp. Clin. Pathol. Dec 2010.19 (6) p. 547-552.

7. Jacobsen,S., Niewold T.A., Kornalijnslijper, E., Toussaint M.J.M. and Gruyi E. 2005

Kinetics of local and systemic isoforms of serum amyloid A in bovine mastitic milk. Vet.

Immunol. Immunopathol.104, p 389-399

8. Janzekovic, M., Brus, M., Mursec, B., Vinis, P., Stajnko, D. and Cus F., (2009). Mastitis

detection based on electric conductivity of milk. J. Achievements in Materials and

Manufacturing Engg. 34 (1)

9. Lahtolainen, T., Rontved, C. and Pyrola, S. 2004 Serum amyloid A and TNFα in serum

and milk during experimental endotoxin mastitis. Vet. Res. 35 p 651-659

10. Larsen, T., 2005. Determination of lactate dehydrogenase (LDH) activity in milk by a

fluorometric assay. J. Dairy Res. 72, 209.

11. Mohammadian, B., 2011. The effect of subclinical mastitis on lactate dehydrogenase in

dairy cows International J. of Anim. and Vet. Advances 3(3): 161-163

12. Mollenhorst, H.; van der Tol, P.P.J.; Hogeveen, H. Somatic cell count assessment at the

quarter or cow milking level. J. Dairy Sci. 2010, 93, 3358-3364.





13. Oviedo- Boyso, J., Valdez- Alarcon, J.J., Cajero- Juarez, M., and Baizabal-Auirre,

V.M.(2007) Innate immune response of bovine mammary gland to pathogenic bacteria,

responsible for mastitis. J.Infect., 54(4):399-409

14. Petersen, H. H., Gardner I.A., Rossito, P., Larsen H.D. Heegard, P.M.H.( 2005) Milk

amyloid A concentration and somatic cell count in diagnosis of bovine mastitis. Mastitis

in Dairy production Current knowledge and future solutions.

15. Pyrola , S. 2003, Indicators of inflammation in the diagnosis of mastitis. Vet. Res. 34 p :

565–578

16. Sharif, A., Ahamad T., Bilal M.Q., Yusuf ,A. and Muhammad,G. 2007. Effect of severiety

of subclinical mastitis on somatic cell count and lactose content of buffalo milk. Pak.Vet.

J., 27(3) 142-144

17. Solverod L., Simonsen, S., Waldmann, A. and Ropstad, E.(2005) Variation in somatic

cell count and milk components in fraction collected quarter milk samples. Mastitis in

Dairy production Current knowledge and future solutions.

18. Viguier, C., Arora, S., Gilmartin, N., Welbeck, K., O'Kennedy, R., 2009. Mastitis

detection: Current trends and future perspectives. Trends Biotechnol. 27,486.

19. Winter, P., Fuch, K. and Schilcher, F. 2005. Host response reaction in lactating ovine

udder during experimental challenge with Staphylococcus epidermidis. Mastitis in Dairy

production Current knowledge and future solutions





Cells of Immune System

Mr. Shaikh Rajesh Ali

Assistant professor, Dept. of Microbiology,

Acharya Prafulla Chandra College, New Barrackpore, Kol-131

The lymphocytes are the cells that define the adaptive immune system as they are the only with

the attributes of specificity, diversity, memory, and self/non-self discrimination. Lymphocytes are

part of group of cells collectively known as white blood cells or leukocytes that participate in the

immune response. Leukocytes, together with the red blood cells (erythrocytes) and platelets,

are the blood cells. Hematopoiesis is the process of formation and differentiation of blood cells.

All blood cells arise from a type of cell called the hematopoietic stem cell (HSC). Stem cells

differentiate into one or more cell types (i.e., they can be unipotent or pluripotent), and have the

property of selfrenewing. In humans, hematopoiesis begins in the embryonic yolk sac in the first

weeks of development. In the third month of development, HSC migrate from the yolk sac to

fetal liver and then to the spleen. These two organs are the responsible of hematopoiesis from

the 3rd to the 7th month of development. After that, the bone marrow takes the place as the

hematopoietic organ.

Early in hematopoiesis, a HSC differentiate into one of two pathways, giving rise to a lymphoid

stem cell or a myeloid stem cell. The lymphoid stem cell gives rise to B, T and NK cells through

the differentiation of progenitor cells specific for each lineage.

The myeloid stem cell generates progenitors for the red blood cells, the platelets and for all the

other leukocytes, which are the granulocytic cells (neutrophils, eosinophils, basophils),

monocytes, mast cells, and dendritic cells.

Differentiation of progenitors into specialized cells is controlled by hematopoietic cytokines or

growth factors. A family of cytokines known as colony-stimulating factors (CSF) is important to

induce the differentiation of the different hematopoietic cell lines. There are different types of





CSFs: multilineage CSF or interleukin-3, macrophage CSF, granulocyte CSF, granulocyte-

macrophage CSF.

The cytokine that regulates the production of red blood cells is from a different family and is

called erythropoietin. Thus, the commitment of a progenitor to a particular lineage depends on

the expression of specific receptors on the cell membrane for particular cytokines.





Hematopoiesis is exquisitely regulated to maintain a constant number of cells in a given time.

This is accomplished by controlling the expression of cytokines and cytokine receptors, and by a

process known as programmed cell death, in which the cell actively participate in its own death.

The morphological changes seen in programmed cell death are known as apoptosis.

The use of HSCs has proved to be an important tool in medicine. Transplantation of HSCs is

used in patients with genetically immunodeficiencies to provide a functional immune system.

Also, in some genetically determined anemias, transplantation of HSC is use to replace the non-

functional red blood cells progenitors. More common is perhaps its use in patients with leukemia

or in patients subjected to chemotherapy and radiation, which have destroyed the hematopoietic

system.

An effective immune response involves two major groups of cells: T lymphocytes and antigen-

presenting cells. Lymphocytes are one of many types of white blood cells produced in the bone

marrow by the process of hematopoiesis. Lymphocytes leave the bone marrow, circulate in the

blood and lymphatic systems, and reside in various lymphoid organs. Because they produce

and display antigen binding cell-surface receptors, lymphocytes mediate the defining

immunologic attributes of specificity, diversity, memory, and self/nonself recognition. The two

major populations of lymphocytes are B lymphocytes (B cells) and T lymphocytes (T cells).

B Lymphocytes:

B lymphocytes mature within the bone marrow; when they leave it, each expresses a unique

antigen-binding receptor

on its membrane. This

antigen-binding or B-cell

receptor is a membrane-

bound antibody

molecule. Antibodies are

glycoproteins that consist





of two identical heavy polypeptide chains and two identical light polypeptide chains. Each heavy

chain is joined with a light chain by disulfide bonds, and additional disulfide bonds hold the two

pairs together. The amino-terminal ends of the pairs of heavy and light chains form a cleft within

which antigen binds. When a naive B cell (one that has not previously encountered antigen) first

encounters the antigen that matches its membrane bound antibody, the binding of the antigen to

the antibody causes the cell to divide rapidly; its progeny differentiate into memory B cells and

effector B cells called plasma cells. Memory B cells have a longer life span than naive cells, and

they express the same membrane-bound antibody as their parent B cell. Plasma cells produce

the antibody in a form that can be secreted and have little or no membrane-bound antibody.

Although plasma cells live for only a few days, they secrete enormous amounts of antibody

during this time. It has been estimated that a single plasma cell can secrete more than 2000

molecules of antibody per second. Secreted antibodies are the major effector molecules of

humoral immunity.

T Lymphocytes:

T lymphocytes also arise in the bone marrow. Unlike B cells, which mature within the bone

marrow, T cells migrate to the thymus gland to mature. During its maturation within the thymus,

the T cell comes to express a unique antigen-binding molecule, called the T-cell receptor, on its

membrane.Unlike membrane-bound antibodies on B cells,which can recognize antigen alone,

T-cell receptors can recognize only antigen that is bound to cell-membrane proteins called

major histocompatibility complex (MHC) molecules. MHC molecules that function in this

recognition event,which is termed “antigen presentation,” are polymorphic (genetically diverse)

glycoproteins found on cell membranes. There are two major types of MHC molecules: Class I

MHC molecules, which are expressed by nearly all nucleated cells of vertebrate species, consist

of a heavy chain linked to a small invariant protein called β2-microglobulin. Class II MHC

molecules, which consist of an alpha and a beta glycoprotein chain, are expressed only by

antigen-presenting cells.When a naive T cell encounters antigen combined with a MHC

molecule on a cell, the T cell proliferates and differentiates into memory T cells and various

effector T cells. There are two well-defined subpopulations of T cells: T helper (TH) and T

cytotoxic (TC) cells.Although a third type of T cell, called a T suppressor (TS) cell, has been





postulated, recent evidence suggests that it may

not be distinct from TH and TC subpopulations. T

helper and T cytotoxic cells can be distinguished

from one another by the presence of either CD4

or CD8 membrane glycoproteins on their

surfaces. T cells displaying CD4 generally

function as TH cells, whereas those displaying

CD8 generally function as TC cells. After a TH

cell recognizes and interacts with an antigen–

MHC class II molecule complex, the cell is

activated—it becomes an effector cell that

secretes various growth factors known collectively as cytokines. The secreted cytokines play an

important role in activating B cells, TC cells, macrophages, and various other cells that

participate in the immune response.

Differences in the pattern of cytokines produced by activated TH cells result in different types of

immune response. Under the influence of TH-derived cytokines, a TC cell that recognizes an

antigen–MHC class I molecule complex proliferates and differentiates into an effector cell called

a cytotoxic T lymphocyte (CTL). In contrast to the TC cell, the CTL generally does not secrete

many cytokines and instead exhibits cell-killing or cytotoxic activity. The CTL has a vital function

in monitoring the cells of the body and eliminating any that display antigen, such as virus-

infected cells, tumor cells, and cells of a foreign tissue graft. Cells that display foreign antigen

complexed with a class I MHC molecules are called altered self-cells; these are targets of CTLs.

An Antigen-Presenting Cell (APC):

An antigen-presenting cell (APC) is a cell that displays foreign antigen complexes with MHC on

its surface. T-cells may recognize this complex using their T-cell receptor (TCR). Although

almost every cell in the body is technically an APC, since it can present antigen to CD8+ T cells

via MHC class I molecules, the term is often limited to those specialized cells that can prime T

cells (i.e., activate a naive T cell). These cells generally express MHC class II as well as MHC





class I molecules, and can stimulate CD4+ ("helper") T cells as well as CD8+ ("cytotoxic") T

cells. To help distinguish between the two types of APCs, those that express MHC class II

molecules are often called professional antigen-presenting cells.

The APCs are very efficient at

phagocytosis, which allows them to present

exogenous as well as internal antigens.

After dendritic cells or macrophages

swallow pathogens, they usually migrate to

the lymph nodes, where most T cells are.

They do this chemotactically: chemokines

that flow in the blood and lymph vessels

"draw" the APCs to the lymph nodes.

During the migration, DCs or macrophages

undergo a process of maturation, like they lose most of their ability to further swallow

pathogens, and they develop an increased ability to communicate with T cells. Enzymes within

the cell digest the swallowed pathogen into smaller pieces containing epitopes, which are then

presented to T cells using MHC class II. After interaction with TCR an additional co-stimulatory

signal is then produced by antigen presenting cell, leading to activation of TH cell and release

different types of cytokines.

Natural Killer (NK) Cell:

A small group of lymphocytes, call null cell, in the peripheral blood do not express the

membrane molecules and receptors that distinguish T and B cell lineage. These cells fail to

produce immunoglobulin. Because these cells do not produce any antigen binding receptor

(ABC receptor), they lack precise immunologic specificity and memory. Most members of null

cell population are large, granular lymphocyte called natural killer (NK) cell.





NK cell were shown to play an important role

in host defense against tumor cell and

against some virus infected cell. NK cell can

recognize the potential target in two different

ways. In some cases, an NK cell employs

NK cell receptors to distinguish

abnormalities, which recognize the tumor

cell or virus infected cell by abnormal cell’s

MHC class I molecules. Another way in

which NK cell recognize potential target cells depends upon the fact that the tumor cell or virus

infected cell display antigen against which immune system produce anti-tumor or antiviral

antibodies and which bind to the abnormal cell surface. Because NK cell express CD16, a

membrane receptor for the carboxyl-terminal end of IgG molecule, called Fc region, they can

attached to these antibodies and subsequently destroy the target cell. This process of cell

destruction is called antibody dependent cell mediated cytotoxicity (ADCC).





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or

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Selected ones will be published in

our next issue of Sept-Oct 2014.

Thanks,

Microbiology World Team



Microbiology World Issue 6

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