Microbiology World Issue 8 Nov Dec 2014 ISSN 2350 - 8774microbiologyworld.com/wp-content/uploads/2014/12/... · The criteria for a microorganism to act as an ideal indicator of pathogens

Microbiology World Issue 8 Nov – Dec 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. Hasnain Nangyal

M.Phil.

Department of Botany, Hazara University, Pakistan

Mr. Sunil Pandey

ELSEVIER Student Ambassador South Asia 2014

B.Sc. Medical Microbiology

Nobel Medical College, Nepal









Table of Content

Page No.

Microbial Indicators: Role in monitoring

of drinking water quality and health prospects 5-8

Prevalence and prevention of thalassemia

in Khyber-Pakhtunkhwa, Pakistan 9-11

Do you have cold? Fomites may be responsible 12-13

Chinese Salt 14-19

Prions: The Killer Proteins 20-28

Hybridoma Technology: A Tool in immunotherapy 29-37

Lyophilisation: A Method of Preserving Biologicals 38-43

Rapid diagnosis of acute respiratory infections by

multiplex endpoint PCR technology 44-47

Bioweapons: A new trend in emerging Sciences 48-58





Microbial Indicators: Role in monitoring of

drinking water quality and health prospects

Gaurav Saxena, Rohan Kanaujia and Ram Naresh Bharagava*

Department of Environmental Microbiology (DEM), School for Environmental Sciences

(SES), Babasaheb Bhimrao Ambedkar University (A Central University), Vidya Vihar,

Raebareli Road, Lucknow 226 025 (U.P.), India

Correspondence E-mail Address: [email protected]

Ensuring the safety of drinking water is an ongoing process. Water that looks perfectly

transparent and clean may be contaminated with pathogens, which may cause serious health

hazards. A clean and safe drinking water supply may be the norm in European and American

countries, but in developing countries, the assessment of clean water is not the rule and

therefore, the waterborne illness outbreaks are common. It is reported that around two and a

half billion people have no access of improved sanitation and more than 1.5 million children die

each year from diarrheal diseases (Fenwick 2006).

A number of disease occur due to contamination of drinking water with urban sewage, feces of

infected humans and animals having different kinds of microorganisms, which are termed as

enteric pathogens. However, the detail information on waterborne pathogens can be found in

Cabral (2010). These include enteric bacteria, viruses and protozoa’s and can be transmitted by

exposure to domestic waste either through swimming in contaminated water, ingesting

contaminated water or eating vegetables that has been irrigated with contaminated water or

grown in contaminated soil (WHO 2008). In general, any practice that involves the application of

domestic wastewater in soil has potential to cause microbial contamination of ground water

since treatment processes applied do not completely remove/inactivate microbial pathogens in

wastewater (Figueras & Borrego 2010).

The examination of drinking water for the presence of indicator microorganisms is a key to

determine the microbiological quality and public health safety because their presence indicate

the potential faecal contamination of water with pathogens and an index of quality deterioration.





Indicator microorganisms are generally not themselves human pathogens (Verhille 2013). This

has been the foundation upon which the protection of public health from waterborne diseases

has been developed. The most widely used indicator microorganisms are coliforms (total

coliforms), faecal or thermo-tolerant coliforms, Escherichia coli, enterococci (Faecal streptococci

or Intestinal enterococci) and bacteriophages. Further, the detail information on microbial

indicators can be found in Cabral (2010) and Figueras & Borrego (2010) (Fig. 1).

Fig. 1: Microbial indicators in drinking water: Total coliform, faecal coliform and E. coli

Adapted from (Verhille 2013)

The criteria for a microorganism to act as an ideal indicator of pathogens in water environment

are as follows (Cabral 2010):

a. The organism should be present whenever enteric pathogens are present and absent

whenever pathogens are absent.

b. The organism present at a density that has some constant direct relationship to the

density of pathogens.

c. The organism should be useful for the analysis of all types of contaminated

environmental waters.

d. The organism should not grow in contaminated water.

e. The organism should be non-pathogenic to humans and easily identifiable.





f. The organism should survive in environment as long as possible.

g. The organism should be the member of intestinal micro-flora of warm-blooded animals.

h. The organism should exist in high number in human intestine as well as in feces.

i. The organism should be detectable by easy, rapid and specific/sensitive, but

economically viable methods

In addition, the potential application of an indicator microorganism should be to indicate:

a. The fecal pollution.

b. The presence of domestic sewage.

c. The presence of microbial pathogens.

d. The efficiency of a particular water or wastewater treatment process.

e. The environmental fate of a target pathogen.

However, whether these indicator bacteria are the suitable indicator of human pathogens, it has

been questioned because of some serious limitations they include (Girones et al., 2010):

a. Sensitive to inactivation through wastewater treatment processes and sunlight exposure.

b. Short survival period as compared to microbial pathogens.

c. Not indicative of exclusive faecal source.

d. Ability to multiply in some natural environments.

e. Inability to recognize faecal contamination source (point or non-point).

f. Less correlation with pathogenic presence.

Further, it is also reported that the drinking water illness outbreaks have occurred both in

presence or absence of indicator microorganisms and involved pathogenic microorganisms that

have contaminated the drinking water and that either were not eliminated during the treatment

process or later failed at the time of outbreak (Figueras and Borrego 2010). These outbreaks

occurred despite the specific legislations that have been designed to prevent them (Figueras

and Borrego 2010).

The World Health Organization (WHO) is an active body in this field and has already published

many guidelines/documents in collaboration with International Water Association (IWA) and





“Organization for Economic Co-operation and Development (OECD)” for improvement in

drinking water quality (WHO 2008). Water safety plan is the most recent that ensure the safety

of drinking water and can be found in the WHO technical guidance document and also available

in WHO directory.

Nevertheless, the presence of indicator microorganisms will likely continue to be used as a

criterion of drinking water quality. Although, there is no any perfect indicator microorganism and

research is ongoing to find a suitable microorganism that can act as a better indicator for the

presence of waterborne pathogens.

Acknowledgement

The authors are highly grateful to University Grants Commission (UGC), Government of India

(GOI) New Delhi for financial support as “Start-Up grant” for this work and UGC Fellowship

received by Mr. Gaurav Saxena is also duly acknowledged.

References

1. Cabral, J. P. S. (2010) Water Microbiology. Bacterial Pathogens and Water. Int. J.

Environ. Res. Public Health 7, 3657-3703.

2. Fenwick, A. (2006) Waterborne Diseases: could they be consigned to history. Science

313, 1077-1081.

3. Figueras, M. J., & Borrego J. J. (2010) New Perspectives in Monitoring Drinking Water

Microbial Quality. Int. J. Environ. Res. Public Health 7, 4179-4202.

4. Girones, R., Ferrus, M. A., Alonso, J. L., Manzano, J. R., Calgua, B., Correa, A. D. A.,

Hundesa, A., Carratala, A., & Bofill-Mas, S. (2010) Molecular detection of pathogens in

water- The pros and cons of molecular techniques. Water Res. 44, 4325-4339.

5. Verhille, S. (2013) Understanding microbial indicators for drinking water assessment:

interpretation of test results and public health significance. National collaborating centre

for environmental health. 1-12.

6. World Health Organization. (2008) Guidelines for drinking water quality. 3rd edn. (1)

Geneva, Switzerland.





Prevalence and prevention of thalassemia in

Khyber-Pakhtunkhwa, Pakistan

1Maria Zubair, 2Tahir Hussain

1Peshawar Medical and Dental College, Warsak Road Peshawar, Khyber-

Pakhtunkhwa, Pakistan

2Atta-ur-Rahman School of Applied Biosciences, National University of Sciences and

Technology, Islamabad, Pakistan

Abstract:

Thalassemia is an inherited autosomal recessive blood disorder characterized by formation of

abnormal haemoglobin which in turn leads to destruction of RBCs. Since it is a recessive trait

and can be controlled, if planned properly, but because of lack of awareness and certain

traditional and cultural practices thalassemiac babies are still reported from rural population of

Khyber-Pakhtunkhwa province Pakistan. This report discusses the prevalence and various

treatment options available for thalassemia patients in a treatment center at Khyber-

Pakhtunkhwa, Pakistan.

Introduction:

Thalassemia is a form of inherited blood disorders, caused by weakening and destruction of

RBCs (Red Blood Cells). The genetic determinants for thalassemia are present on autosomes

and the condition arises when the genes are missing or variant of the genes are present. This

causes the malfunction of the body to make normal haemoglobin (Hb). Life span of Red Blood

cells (RBC) with normal haemoglobin (Hb) is 120 days while in thalassemia it becomes 10 to 15

days only.

Normally the majority of adult hemoglobin <Hb A> is composed of four protein chains i.e; two

alpha and two beta globin chains arranged in a hetrotetramer form. In thalassemia, patients

have defect either in alpha or beta globin chain causing production of abnormal RBC.





Common treatment options available in Pakistan:

1. Blood transfusion: Blood transfusion is a common option available for extending life

expectancy of thalassemiacs. Since thalassemia patients need regular blood transfusions to

sustain their life and multiple transfusions can result in iron overload which then results in

severe physiological dysfunctions in the body.

2. Bone marrow transplantation: Bone marrow transplantation offers about 70% success rates in

pakistan1. However it is an expensive surgery and costs about Rs. 30 lac in Pakistan1.

3. Hydroxy urea: This drug is generally used in treatment of leukemia, breast cancer and

malignancy. However, recently, this drug was found to be effective against thalassemia as well1.

According to the research, about 60% of patients didn’t need further blood transfusion while in

remaining 40% there was decreased rate of blood transfusion noticed1.

As a case study in Hamza foundation Peshawar, a welfare organization providing healthcare

services to thalassemiac children, exercised the hydroxy urea treatment strategy on about 160

patients, out of them 31 patients are not receiving any further blood transfusion from past 9

months1. However certain side effects of this drug, such as, leukocytopenia and bone marrow

depression were noticed1.

Prevalence: About 823 thalassemiac patients from all over Khyber-Pakhtunkhwa, Pakistan,

registered at Hamza Foundation in seven years period1. These figures are available only on our

record. Of course there could be many more thalassemiac patients struggling for their life. 95%

of the thalassemiac patients belong to poor, deserving and uneducated class1. It is a

troublesome disease common in children and young patients with extremely high mortality rate.

Prevention: Prevention could be a better alternative as compared to expensive treatment.

Since thalassemia is a hereditary condition so controlling thalassemia in the population will only

be possible if thalassemia free babies are delivered. For this reason we need to take certain

measures in order to prevent thalassemia in the coming generations.

1. Family marriages: Cousin marriages are quite common in villages and poor set up. For

this purpose government should play its role in educating people about the hazards

resulting from cousin marriages.





2. Pre marriage screening: All the first and second relatives of known thalassemia patients

should be screened. If they are diagnosed with diseased or carriers, their record should

be kept in view so that they must not marry member from other thalassemiac family.

3. Prenatal screening: If a thalassemiac couple gets expected they must undergo prenatal

screening i.e; CVS (Chorionic Villus Sampling) during first trimester. If the fetus is

diagnosed as thalassemiac major then it must be aborted. Healthcare authorities and

ethical bodies agree all over the world that aborting a fetus in certain conditions like for

saving life of mother or if fetus is diagnosed to b abnormal is not illegal.

4. Spreading the awareness: Generally uneducated people in our population do not know

the basic information regarding thalassemia. They should be properly educated about

the causes and ways to prevent thalassemia. Awareness campaign should be launched

through both electronic and print media. Radio, Tele vision, daily newspapers,

magazines, distributing pamphlets, conducting seminars, guiding imam masjids,

chodries and khans to educate people in their communities.

Acknowledgement:

We are grateful to Dr. Tariq khan, Hamza Foundation, for providing data about thalassemia

patients.

References:

Hamza Foundation, 2-A Park Avenue, University Town Peshawar, Khyber-Pakhtunkhwa

Pakistan.





Do you have cold? Fomites may be responsible

Oyediji Kehinde Eyitayo

University of Abuja, Abuja, Nigeria

The common cold is a viral infectious disease of the upper respiratory tract which primarily

affects the nose. A cold usually begins with fatigue, a feeling of being chilled, sneezing and a

headache, followed in a couple of days by a runny nose and cough, nasal congestion and a

sore or scratchy throat, sometimes accompanied by loss of appetite, sneezing, hoarseness,

watery eyes, low-grade fever, headache, and body aches. Symptoms may begin within 16 hours

of exposure and typically peak two to four days after onset. The symptoms of the common cold

will typically last anywhere from 4-14 days, with most individuals improving in one week. The

common cold is the most common human disease and all peoples globally are affected. The

common cold is generally mild and self-limiting with most symptoms generally improving in a

week.

The common cold virus is typically transmitted via airborne droplets (aerosols), direct contact

with infected nasal secretions, or fomites. Diseases that spread by droplet transmission, fecal–

oral transmission, or contact transmission often do so by means of fomites. Germs commonly

live on fomites for minutes or hours or sometimes even longer.

Fomites are inanimate objects that can carry pathogenic agents from one susceptible source to

another. These objects can be anything, such as a handset, door knobs (handle), laboratory

benches, laptop keypads, ATM machines, money, clothes, dishes, books, pens, silverware, or

escalator hand rail. A cold virus can live on objects for several hours and can thus be acquired

from contact with these objects. Fomite transmission works best on hard, durable surfaces like

dishes, books, door knobs, telephones, and hand rails, all of which people come in contact with

on a daily basis.

Regular hand washing appears to be effective in reducing the transmission of cold viruses. Not

touching the nose or eyes is another. Individuals with colds should always sneeze or cough into





a facial tissue, and promptly throw it away. If possible, one should avoid close, prolonged

exposure to persons who have colds.

In the laboratory and other healthcare environments, gowns and disposable gloves are also

used. Isolation, e.g. quarantine, is not possible as the disease is so widespread and symptoms

are non-specific. Vaccination has proved difficult as over 200 different viral types are associated

with colds and they mutate rapidly. Creation of a broadly effective vaccine is thus highly

improbable. Zinc supplements may help to reduce the prevalence of colds. Routine vitamin C

supplements do not reduce the risk or severity of the common cold, though they may reduce its

duration. Because common cold viruses can survive up to three hours outside the nasal

passages on inanimate objects and skin, cleaning environmental surfaces with a virus-killing

disinfectant might help prevent spread of infection.

No medications or herbal remedies have been conclusively demonstrated to shorten the

duration of infection. Treatment thus comprises symptomatic relief. Getting plenty of rest,

drinking fluids to maintain hydration, and gargling with warm salt water, are reasonable

conservative measures. One study has found chest vapor rub to provide some relief of

nocturnal cough, congestion, and sleep difficulty. Treatments that help alleviate symptoms

include simple analgesics and antipyretics such as ibuprofen and acetaminophen/paracetamol.

Other decongestants such as pseudoephedrine are also effective in adults. Ipratropium nasal

spray may reduce the symptoms of a runny nose but has little effect on stuffiness. Much of the

benefit from treatment is however attributed to the placebo effect.

Antibiotics have no effect against viral infections and thus have no effect against the viruses that

cause the common cold. Due to their side effects antibiotics cause overall harm, but are still

frequently prescribed. Some of the reasons that antibiotics are so commonly prescribed include

people's expectations for them, physicians' desire to help, and the difficulty in excluding

complications that may be amenable to antibiotics. There are no effective antiviral drugs for the

common cold even though some preliminary research has shown benefits.





Chinese Salt 1Ammara Nawaz, 2Hasnain Nangyal, 3Sikander Khan Sherwani,

4Noor Nasir Khattak

1Department of Zoology Punjab University

2Department of Botany Hazara University Mansehra Khyber Pakhtoonkhwa

3Department of Microbiology Fedral Urdu University of Arts Science & Technology

Karachi

4Assistant Professor Gynecology and Obstetrics Kohat Medical College Khyber Medical

University

To say the history of salt is essentially the history of the world is not an overstatement. Some

call salt a "primordial condiment," and rightfully so. It has been part of this earth for as long as

there has been water and rock to create it. It has been a highly-valued commodity that served

many purposes, but perhaps the most useful and powerful purpose is preservation. Salt has an

uncanny ability to preserve just about anything, a vegetable or even a human cadaver. In the

history some riots for salt are also been observed.

Several types of salts are used by man in foods and for different applications also. Some of

them are Iodized table salt, table salt, sea salt, kosher salt, Chinese salt, pickling salt, black salt,

Himalayan pink salt, flake salt, smoked salt, seasoned salt, Hawaiian salt and many more.

Chinese salt is now a days a widely used salt in dishes. It is a white crystalline substance the

sodium salt of glutamic acid that has little flavour itself but enhancesthe flavour of proteins either

by increasing the amount of saliva produced in the mouth or by stimulating the taste buds used

as a food additive especially in Chinese Formula: NaC5H8O4. It is also called sodium glutamate

with abbreviation MSG.

History of Chinese salt

First stage of the story begins in 1908 with chemist Ikeda Kikunae’s isolated this ingredient in

sea kelp that gave flavor to konbu dashi, the standard Japanese broth. The product that





emerged from Ikeda’s laboratory, Monosodium glutamate was quickly patented in Japan, the

United States, England, and France. In domestic announcements of his invention, Ikeda

proposed calling its distinctive taste umami a term derived from the word in Japanese meaning

“tasty.” Ikeda brought the powdered substance to iodine manufacturer Suzuki Saburo, whose

Suzuki Chemical Company began marketing it in 1909 under the brand name Ajinomoto,

meaning “essence of taste.”

Fig. 1: Varieties of salts used by man.

What are glutamates?

Glutamate is one of the most general amino acids found in nature. It is the main component of

many proteins and peptides, and is present in most tissues. Glutamate is also produced in the

body and plays an essential role in human metabolism. Virtually every food contains glutamate.

It is a major component of most natural protein foods such as meat, fish, milk and some

vegetables. This amino acid has a flavor enhancing property in both free and combined state. It





is a major component of MSG, so when MSG is added into any food its flavor is enhanced due

to presence of glutamate in it giving food an Umami flavor which is a meaty taste.

MSG and our Metabolism

Our body is capable to digest MSG as it is capable to digest the naturally present amino acid

glutamate. Our body metabolizes glutamate easily. Once glutamate has entered into the body of

a human it metabolizes it and can’t distinguish between the origins of the amino acid. If it is a

part of a tomato or MSG added into the food.

MSG is low in sodium

It is a popular belief that MSG contains high percentage of sodium ions .Actually MSG contains

only one third the amount of sodium as compared to table salt. So it is safe to use by high blood

pressure patients, heart patients and many more that can’t afford much sodium in their diet and

also put a little effect on palatability even giving food a meaty more pleasant taste.

Contradictory views about Chinese salt

Many contradictory statements are there related to Chinese salt. Many people even scientists

think that Chinese salt be not good for human health and has some health risks associated to it.

Several aspects of Chinese salt in relation to human health are studied in detail. Some of these

are discussed as below:

Chinese salt and pregnant women

It was once thought that Chinese salt in not good for pregnant females, lactating mothers and

the fetus or placenta. Now various studies have proved that MSG intake has no adverse effect

in all above cases. It is known through deep investigations that the ratio of MSG in the blood

cannot be increased by the intake (Chinese salt). Pitkin a scientist to see the effect of MSG

increase on pregnant females and fetus conducted an experiment. He injected high amount of

MSG in the blood of the Monkey pregnant female and noticed no increase in level of MSG blood

level up to a dose of 220mg\kg of maternal weight. Thus no raise in level of blood MSG and no

effect of it on the female and the fetus were concluded.





Chinese salt and lactating mothers

No bad effect of Chinese salt was seen in lactating mothers too. By certain experiments this

was proved. Lactating women were given MSG at about 100mg\kg of body weight and its effect

was noticed on the mother and the infant. No increase of level of glutamate in human milk was

seen and no effect was observed on the infant also. Naturally human milk has 10 times more

ratio of glutamate in it as compared to cow’s milk. Thus MSG intake in diet was declared not a

risk for the infants feeding on breast milk.

Chinese salt and its neurological effects

In the brain, glutamate serves as a neurotransmitter in addition to its general role in protein and

energy metabolism. Concerns were raised in the late 1960s by John Olney, M.D., of

Washington University, that high doses of MSG may adversely affect brain function. He

examined the possibility of MSG-induced brain lesions through injection or force-feeding

methods in rodents. But the dosage used in this experiment were very high not comparable to

the amount of glutamate taken up by a man by using Chinese salt in his diet in normal days.

Studies say that an amount of 40g\kg weight of the man of MSG is even safe which is actually

5000 times then normally taken amount by a man.

In another experiment by Bazzano it was also concluded that MSG has no bad effect on

humans. In this experiment 11 humans were given 147g\day of MSG for about 42 days and no

side effects were observed in them.

William Pardridge, M.D pointed up that dietary glutamate does not enter the brain because the

blood-brain barrier maintains a transport system for acidic amino acids, such as glutamate, to

effectively exclude circulating glutamate from the brain. Pardridge also showed that the levels of

brain glutamate do not rise or fall with changes in plasma glutamate levels. This point was

strengthened by many other investigations by many scientists.





Thus it was concluded that ordinary daily intake level of MSG has no bad effect on human brain

and nervous system.

Chinese salt and hypersensitive reactions

Many people are observed by scientists having allergy to some food products or substance

such as cheese or peanuts etc due to many reasons. Some scientists questioned if Chinese salt

or MSG may be allergic to some people. Several investigations were made to study this

hypothesis.

In 1991, after reviewing the literature on MSG and food allergy and safety, a panel of the

American College of Allergy, Asthma and Immunology concluded that MSG is not an allergen

and reaffirmed its safety as a food ingredient.45. More recently, Ronald Simon, M.D.,

department of allergy and immunology, Scripps Clinic, La Jolla, California, conducted a well-

designed, double-blind, placebo-controlled study of 65 subjects with chronic urticaria. None of

the subjects exhibited positive reactions to doses of 2.5 g of MSG.

Chinese salt in relation to children health

It has been speculated that children would metabolize oral MSG more slowly than adults.

However, research conducted by Stegink and colleagues at the University of Iowa showed that

children as young as one year old metabolize glutamate as effectively as adults.

Chinese salt and Asthma

Early poor studies said that MSG might be a cause of asthma. However further controlled

investigations pointed out that it is not the case. MSG was not a cause of asthma. In an

experiment humans were given 25g\kg weight of MSG per day for a few days. No difference in

pulmonary reactions was noted in these humans.

In 1991 and 1993, researchers from the National Institutes of Health’s Institute of Allergy and

Infectious Diseases presented data analyzing the possible association of MSG to asthma. In

one study, they challenged 13 non-asthmatics and 30 asthmatics with a total dose of 7.6 g of





MSG. Upon observation, none of the non-asthmatics experienced any change in pulmonary

reactions and only one asthmatic participant experienced some discomfort.

Chinese Restaurant Syndrome (CRS)

In 1968, Robert Ho Man Kwok, M.D., described a collection of symptoms he allegedly

experienced after eating Chinese food. He coined the phrase “Chinese Restaurant Syndrome”

(CRS) to describe these symptoms, which included numbness at the back of the neck and a

feeling of pressure in the face and upper chest muscles. As a consequence of Kwok’s account,

Kerr and colleagues developed a subjective questionnaire to assess the prevalence of CRS in

the population. The survey employed listed 18 adverse symptoms related to food, of which three

were related to CRS. Of the 3,222 general households that responded to the survey, 43 percent

reported food-related adverse reactions, but only 1.8 percent reported possible CRS symptoms.

Richard Kenney, M.D., of George Washington University did a lot of experiments to evaluate the

use of MSG and relation of CRS. He tested 60 people with He further tested 60 subjects with

orange juice, spiced tomato juice, black coffee, flavored milk and a two percent MSG solution.

Upon examining reactions, Kenney found that six subjects responded to coffee, six to spiced

tomato juice and only two to the MSG solution, indicating that MSG was not unique in producing

symptoms typical of CRS. However several people in many experimental studies have been

seen showing some indications of CRS by the uptake of MSG. These include tightness in the

chest, flushing and headache.

Conclusion

MSG is one of the most intensely used food ingredient on today’s culinary world. Numerous

researches have been conducted to see its adverse effects on human body and no concrete

evidence has been found to say that MSG is bad for health in the regular amounts used by the

common man. Chinese salt is safe to eat in a limit as it is mostly used. However excess of MSG

may cause bad results on human health.





Prions: The Killer Proteins

Mr. Shaikh Rajesh Ali

Assitant Professor, Dept. Of Microbiology, Acharya Prafulla Chandra College,

New Barrackpore, Kolkata -700131

Introduction:

Prions is an acronym for ‘proteinaceous infectious particles’. The term was coined in 1982 by

Stanley B. Prusiner, a neurologist at the University of California at San Francisco, who proposed

that a new type of pathogen consisting solely of protein is responsible for deadly

neurodegenerative diseases called Transmissible Spongiform Encephalopathies (TSEs) and

received the Nobel Prize in medicine in 1997 for his work on them. These include scrapie in

sheep, bovine spongiform encephalopathy (BSE or ‘mad cow disease’) in cattle and Creutzfeldt-

Jakob Disease (CJD) in people.

Chemical Nature of prions:

The chemical nature of the prions, as stated earlier, is considered to be proteinaceous and they

have no nucleic acids of their own. This has been indicated by the various experimental

evidences gathered so far. This aspect of prion has been investigated by treating them with

nucleases (the enzymes that digest nucleic acids) and proteases (the enzymes that digest

proteins). It has been observed that the nucleases have no effect of prion infectivity, whereas

proteases can drastically reduce a prion infectivity. In addition, prions show high resistance to

ionizing and ultraviolet radiations, which act mainly on nucleic acids.

Structure of Prions:

Prion is 100 times smaller than a virus, contains only protein, are heterogenous in size and

density, and can exist in many molecular forms. The protein that prions are made of (PrP) is

found throughout the body, even in healthy people and animals. However, PrP found in

infectious material has a different structure and is resistant to proteases, the enzymes in the

body that can normally break down proteins.





The normal form of the protein is called PrPC, while the infectious form is called PrPSc — the C

refers to 'cellular' or 'common' PrP, while the Sc refers to 'scrapie', a prion disease occurring in

sheep.

PrPC is a normal protein found on the membranes of cells. It has 209 amino acids (in

humans), one disulfide bond, a molecular weight of 35-36 kDa and a mainly alpha-

helical structure.

The infectious isoform of PrP, known as PrPSc, is able to convert normal PrPC proteins

into the infectious isoform by changing their conformation, or shape; this, in turn, alters

the way the proteins interconnect. Although the exact 3D structure of PrPSc is not known,

it has a higher proportion of β-sheet structure in place of the normal α-helix structure.

Aggregations of these abnormal isoforms form highly structured amyloid fibers, which

accumulate to form plaques.

Synthesis of prion:

The prion is a product of a human gene,

termed the PrP gene, found on

chromosome 20. This gene contains two

exons separated by a single intron.

Exon I and Exon II are transcribed and

the two RNAs ligated into a single

mRNA. This mRNA contains an open

reading frame (ORF) or protein coding region which is translated into the PrP protein. The PrP

protein is a precursor of the prion protein. It is termed PrP 33-35.

The PrP 33-35 undergoes several post-translational events to become the prion protein (PrP

27-30):

a) Glycosylation - at two sites.

b) Formation of a disulfide bond between two cysteine residues.

c) Removal of the N-terminal signal peptide.

d) Removal of the C-terminal hydrophobic segment.





e) Addition of a phosphatidylinositol glycolipid at the C-terminal.

f) Removal of the N-terminal first 57 amino acids.

In normal cells only the PrP 33-35

protein is synthesized. It is found in

the neural cell membrane where it's

function is to sequester Cu++ ions.

In abnormal ("infected") cells, the

PrP 27-30 is produced from the

PrP 33-35 protein. The PrP 27-30

triggers a series of reactions that

produce more PrP 27-30 proteins,

i.e., PrP 27-30 induces its own

synthesis. In addition to the post

translational modifications, the PrP

27-30 protein differs from the PrP 33-35 protein in a single amino acid residue. Residue 178 in

the PrP 27-30 contains an asparagine residue whereas the PrP 33-35 protein has an aspartate

residue at this position. This causes a conformational change in the PrP 27-30 protein from an

a-helix to a b-sheet. This conformational change in the PrP 27-30 protein has three effects:

a) It imparts to the PrP 27-30 protein the ability to induce the same a-helix to b-sheet

conformation in the PrP 33-35 protein. This is a permanent conformational change. It

thus induces its own "replication."

b) The b-sheet-forming peptides aggregate to form amyloid fibrils.

c) The amyloid fibrils kill thalamus neurons through apoptosis, a programmed series of

events that leads to cell death.

Prion replication mechanism:

The hypothesis that tried to explain how prions replicate in a protein-only manner was the

heterodimer model. This model assumed that a single PrPSc molecule binds to a single PrPC





molecule and catalyzes its conversion into PrPSc. The two PrPSc molecules then come apart and

can go on to convert more PrPC.

Transmission of prions:

Spread of the disease is via horizontal transmission, i.e., transmission from one person to

another, either directly or by fomites or by ingestion of contaminated meat. These diseases in

humans are now grouped together according to whether they are sporadic, inherited, or

acquired. Most cases of prion disease are sporadic; that is, they arise spontaneously for no

known reason. More rarely prion disease is inherited due to a faulty gene, or acquired by

medical procedures, transfusions, or contaminated food. Sporadic and inherited prion disease

occurs worldwide in all populations. The incidence of sporadic CJD is around 1 per million of

the population per annum; males and females are equally affected. The incidence of the

various acquired prion diseases, however, is more localized to specific groups and populations.

Prion pathway to brain:

The pathway to the brain is a subject of significant research, but currently, no comprehensive

answer exists. In TSE linked to consuming TSE-infected material, it is theorized that once prions

are ingested, they are taken up by the lymphoid tissue that drains the gastrointestinal tract

including Peyer's Patches and other nodes. Prions also have been found in tonsil, spleen and





appendix. From the lymphatic system, research suggests that prions replicate, access and

move through the autonomic nervous system to the central nervous system. Once in the brain,

the higher concentration of cellular prion protein speeds up the replication process. Prions also

may enter lesions or wounds in the oral cavity and access the vagus nerve as a pathway to the

brain.

Recent laboratory research using fluorescent dye to "brand" scrapie proteins has tracked prions

as they invade nerve cells and then travel along wire-like circuits to points of contact with other

cells. This appears to be the way the prions that cause TSE invade nerve cells and are

transported along neural circuits throughout the nervous system.

Diseases caused by prions:

Affected animal(s) Disease

sheep, goat Scrapie

cattle Bovine spongiform encephalopathy (BSE), mad cow

disease

mink Transmissible mink encephalopathy (TME)





white-tailed deer, elk, mule deer,

moose Chronic wasting disease (CWD)

cat Feline spongiform encephalopathy (FSE)

nyala, oryx, greater kudu Exotic ungulate encephalopathy (EUE)

ostrich Spongiform encephalopathy

(Not been shown to be transmissible.)

human

Creutzfeldt–Jakob disease (CJD)

iatrogenic Creutzfeldt-Jakob disease (iCJD)

variant Creutzfeldt-Jakob disease (vCJD)

familial Creutzfeldt-Jakob disease (fCJD)

sporadic Creutzfeldt-Jakob disease (sCJD)

Gerstmann–Sträussler–Scheinker syndrome (GSS)

Fatal familial insomnia (FFI)

Kuru

A survey of spongiform diseases:

Classic CJD or Creutzfeldt-Jakob disease (human)

The most prevalent of the spongiform diseases

Occurs spontaneously in 1 out of a million people

10% of cases are inherited mutations in the PRPN gene

Usually strikes people age 50 to 75

Symptoms: dementia, muscle twitching, vision problems

Fatal Familial Insomnia (human)

All cases are inherited mutations in the PrP gene

Usually strikes people age 36 to 61





Disruption of sleep/wake cycle leads to coma, then death

Scrapie (goats, sheep)

Occurs as infection in genetically susceptible sheep

There is no evidence of spread to humans

BSE or Bovine Spongiform Encephalopathy (cattle)

Also known as "Mad Cow Disease" because infected animals act strangely and can be

aggressive

Spread rapidly through Britain by rendering

Chronic Wasting Disease (deer, elk)

Infectious disease in wild deer and elk primarily in the western United States

Drooling, difficulty swallowing, weight loss

Kuru (human)

Struck members of the Fore tribe in the 1950s and 1960s

Muscle weakness, loss of coordination, tremors, inappropriate episodes of laughter or

crying

Transmitted by ritual cannibalism as part of funeral ceremonies

Diseases caused by prions are known as

spongiform diseases, because the brain

tissue in infected individuals is filled with

holes, giving it a sponge-like appearance.

Although prions are found throughout the

brain, the symptoms of spongiform diseases

vary according to the regions they are most

concentrated in. There are currently no

effective treatments for spongiform diseases

and all are fatal.





Treatments of prions:

The mechanism of prion replication has implications for designing drugs. Since the incubation

period of prion diseases is so long, an effective drug does not need to eliminate all prions, but

simply needs to slow down the rate of exponential growth. Models predict that the most effective

way to achieve this, using a drug with the lowest possible dose, is to find a drug that binds to

fibril ends and blocks them from growing any further.

Advancements in computer modeling have allowed for scientists to identify compounds which

can serve as a treatment for prion caused diseases, such as one compound found to bind a

cavity in the PrPC and stabilize the conformation, reducing the amount of harmful PrPSc.

Recently, anti-prion antibodies capable of crossing the blood-brain-barrier and targeting

cytosolic prion protein (an otherwise major obstacle in prion therapeutics) have been described.

Possible immunotherapeutic strategies for prion disease

a | Ablation of mature follicular dendritic

cells (FDCs) delays the development of

prion disease in mice. However, treatment

with multiple doses of CpG-containing

oligodeoxynucleotides (CpG ODNs)

produces severe unwanted side-effects,

including immunosuppression, liver

necrosis and thrombocytopaenia. b |

Treatment with the lymphotoxin- receptor

(LT- R)–Ig fusion protein seems to be

better tolerated, but the best protection is

achieved when the fusion protein is

administered immediately after exposure

to prions. c | Vaccination against a self-

protein is difficult because of immune

tolerance, and it has the potential to induce





autoimmune disease. Mice devoid of the prion protein (PrP) develop high PrP-specific antibody

titres after immunization with PrP-derived peptides or full-length PrP; however, tolerance in wild-

type mice allows the induction of only low titres of PrP-specific antibodies. d | Treatment with

dimeric full-length PrP fused to the Fc portion of human IgG1 (PrP–Fc2) delays the development

of prion disease in transgenic mice, most probably owing to its interaction with the disease-

associated PrP (PrPSc). LT- 1 2, LT heterotrimer; TLR, Toll-like receptor.





Hybridoma Technology: A Tool in

immunotherapy

Nwabufo Chukwunonso Kingsley

Department of Biochemistry, Faculty of life sciences, University of Benin, Benin city,

Edo state, Nigeria.

Corresponding Email: [email protected]

Abstract

Hybridoma technology involves forming hybrid cell lines (called hybridomas) by fusing an

antibody -producing B cell with a myeloma (B cell cancer) cell that is selected for its ability to

grow in tissue culture and for an absence of antibody chain synthesis. The antibodies produced

by the hybridoma are all of a single specificity and are therefore monoclonal antibodies. These

monoclonal antibody formed can be purified by antigen affinity chromatography. Considerable

advancements in the last 10-15 years have been made to improve the quality and yield of

monoclonal antibody by hybridoma technology such as the use of myelomas that do not secrete

their own antibodies and that therefore do not interfere with the production of the required

antibody. Immunotherapy is the "treatment of disease by inducing, enhancing or suppressing an

immune response". The quest to develop advanced and effective diagnosis and treatment to

certain disease conditions has led to advancement in immunotherapeutic procedures of which

the use of hybridoma technology has widely been credited to. In this so-called Monoclonal

antibody therapy, monoclonal antibody are targeted against specific antigen and thus enhances

the ability of the immune system to suppress the antigen. Some of the targeted disease

conditions in which monoclonal antibody therapy has widely been used include: cancer

(Tositumomab for non-Hodgkins lymphomas), autoimmune diseases (Infliximab and

Adalimumab which are effective in rheumatoid arthritis, crohn`s disease and uclerative Colitis.

Keywords: Hybridoma technology, Monoclonal antibody, Immunotherapy, Monoclonal Antibody

Therapy.





Introduction

Hybridomas are cells that have been engineered to produce a desired antibody in large

amounts, to produce monoclonal antibodies [1,2]. Monoclonal antibody can be produced by a

technique called hybridoma technology. Hybridoma technology involves forming hybrid cell lines

(called hybridomas) by fusing a specific antibody-producing B- cells with a myeloma(B- cell

cancer) cell that is selected for its ability to grow in tissue culture and for an absence of antibody

chain synthesis. The antibodies produced by the hybridoma are all of single specificity and are

therefore monoclonal antibody [2].

In 1975, Ce`sar Milstein and Georges J. F kohler invented the production of monoclonal

antibodies and they shared the nobel prize of 1984 for medicine and physiology with Niels Kaj

Jerne, who made other contributions to immunology. The term hybridoma was coined by

Leonard Herzenberg during his sabbatical in César Milstein's laboratory in 1976/1977 [1].

Generally, the production of one MAb, using the hybridoma technology, costs between $8,000

and $12,000. The average reasonably SK can generate only 15 to 30 hybridoma fusions per

year, but in an environment where the focus is on diagnostic- or therapeutic-quality MAbs, there

are additional significant limitations than can further decrease throughput [3].

Passive immunity using monoclonal antibody is the largest category of biotechnology developed

chemotherapy. In passive immunity, instead of injecting a specific antigen and inducing the

body to produce an immune response (vaccination), a specific antibody targeted against an

antigen is introduced into the body which enhances the ability of the immune system to

suppress the antigen for example, administering multiple doses of HERCEPTIN, a monoclonal

antibody against breast cancer to a patient diagnosed of breast cancer helps the immune

system to suppress the growth of the cancer cell. This is the basis of the so-called "Monoclonal

Antibody Therapy" which has revolutionalised immunotherapy.

Methodology

Laboratory animals usually mice or rat are injected with an immunizing dose of a specific

antigen. Once the animal is making a good antibody response, the spleen is removed and a cell





suspension is prepared, lymph node cells may also be used. These cells are fused with a

myeloma cell line by the addition of poly ethylene glycol which promotes membrane fusion. The

myeloma cell is a B cell cancer cell and consequently can grow indefinitely. In addition, the

myeloma cell is deficient in the enzyme HYPOXANTHINE GUANINE PHOSPHORIBOSYL

TRANSFERASE (HPGRT).This is a key enzyme in the biosynthesis of purines and pyrimidines

using the salvage pathway. This means that the myeloma cells can only use the De novo

pathway to synthesize purines and pyrimidines but cannot use the salvage pathway. However,

the spleen cells can use both the De novo and salvage pathway to synthesize purines and

pyrimidines. Only small proportion of the cells fuse successfully. The Fusion mixture is then

cultured in a medium containing HYPOXANTHINE, AMINOPTERIN AND THYMIDINE called a

HAT - MEDIUM.

Aminopterin blocks De novo pathway of purine and pyrimidine synthesis. However this pathway

can be bypassed in favour of the salvage pathway by the provision of hpoxanthine and

thymidine in the HAT medium. The spleen cells can grow in the HAT medium using the salvage

pathway but myeloma cells cannot grow in the HAT medium because they are incapable of

using the salvage pathway due to the deficiency of the enzyme HPGRT. They therefore die in

the HAT medium. When the culture was set up in the HAT-medium, it contains spleen cells,

myeloma cells and fused cells. The unfused spleen cells die in culture naturally after 1-2 weeks.

Unfused myeloma cells are killed by the HAT-medium but fused cells survive as they have the

immortality of the myeloma and the ability of spleen cells to use the salvage pathway for purine

and pyrimidine biosynthesis. Some of the fuse cells or hybridoma will also have the antibody

producing capacity of the spleen cells.

Any wells containing growing cells are tested for the production of the desired antibody and if

positive, the cultures are cloned, that is, plated out so that only one cell is in each well. This

produces a clone of cells derived from a single progenitor which is both immortal and produces

monoclonal antibody.





Purification of Monoclonal

Antibodies

Monoclonal antibodies may need

to be purified before they are

used for a variety of purposes.

Before final purification, the

cultures may be subjected to cell

fractionation for enrichment of

the antibody protein. In E. coli,

the antibodies may be secreted

in the periplasm, which may be

used for enrichment of antibody,

so that further purification is

simplified. Alternatively the

antibodies may be purified from

cell homogenate or cell debris

obtained from the medium. (6, 7)

Antibodies can be purified by

anyone of the following techniques

(I) ion-exchange chromatography;

(II) antigen affinity chromatography

Advancements in Hybridoma Technology

Considerable efforts during the last 10-15 years have been made to improve the yield of

monoclonal antibodies using hybridoma technology [4, 5]. These efforts include the following:

(1) The substitution of a chemical fusion promoter (P.E.G.) for the Sendai virus initially used to

promote fusion, and

(2) The use of myelomas that do not secrete their own antibodies and that therefore do not

interfere with the production of the required antibody





(3) A continuous cell line (Sp 2/0) was used as a fusion partner for the antibody producing B

cells.

(4) Feeder layers consisting of extra cells to feed newly formed hybridomas were used for

optimal growth and hybridoma production. Advancements OR Improvements in Hybridoma

Technology – Considerable efforts during the last 10-15 years have been made to improve the

yield of monoclonal antibodies using hybridoma technology. The most common feeder layers

consist of:

• murine peritoneal cells,

• marcrophages derived from mouse, rat or guinea pig

• extra non immunized spleen cells,

• human fibroblasts, human peripheral blood monocytes or thymus cells; these feeder cells had

some limitations like depletion of nutrients meant for hybridoma and contamination, so that other

sources of hybridoma growth factors (HGF) like interleukin-6 (II-6) derived from human cells

were used.

Application of Hybridoma Technology in Immunotherapy

The production of monoclonal antibody by hybridoma technology makes this technology an

indispensable tool in immunotherapy. The quest to develop advanced and effective diagnosis

and treatment to certain disease conditions has led to advancement in immunotherapeutic

procedures of which the use of hybridoma technology has widely been credited to.

Immunotherapy is the "treatment of disease by inducing, enhancing, or suppressing an immune

response" [8]. Immunotherapies designed to elicit or amplify an immune response are classified

as activation immunotherapies, while immunotherapy’s that reduce or suppress are classified as

suppression immunotherapy’s [9]. There are several types of immunotherapy’s including,

monoclonal antibody, and nonspecific immunotherapy and Cancer vaccines [9]. We shall look at

applications of monoclonal antibody in immunotherapy. Immunotherapy developed as a

technique with the discovery of the structure of antibodies and the development of hybridoma

technology, which provided the first reliable source of monoclonal antibodies, and allowed

therapeutic development since the 1970s [10, 11]. These advances allowed for the specific





targeting of tumors both in vitro and in vivo. Initial research on malignant neoplasms found MAb

therapy of limited and generally short-lived success with malignancies of the blood [12, 13].

Furthermore treatment had to be specifically tailored to each individual patient, thus proving to

be impracticable for the routine clinical setting. Throughout the progression of monoclonal drug

development there have been four major antibody types developed: murine, chimeric,

humanized and human [9].

Initial therapeutic antibodies were simple murine analogues, which contributed to the early lack

of success. It has since been shown that these antibodies have: a short half-life in vivo (due to

immune complex formation), limited penetration into tumour sites, and that they inadequately

recruit host effectors functions [14]. To overcome these difficulties the technical issues initially

experienced had to be surpassed. Chimeric and humanized antibodies have generally replaced

murine antibodies in modern therapeutic antibody applications. Hybridoma technology has been

replaced by recombinant DNA technology, transgenic mice and phage display [15].

Understanding of proteomics has proven essential in identifying novel tumour targets. Some of

the targeted disease disease conditions in which monoclonal antibody therapy has widely been

used include:

Cancer

Anti-cancer monoclonal antibodies can be targeted against malignant cells by several

mechanisms:

Radioimmunotherapy (RIT) involves the use of radioactively conjugated murine antibodies

against cellular antigens. Most research currently involved their application to lymphomas, as

these are highly radio-sensitive malignancies. To limit radiation exposure, murine antibodies

were especially chosen, as their high immunogenicity promotes rapid clearance from the body.

Tositumomab is an example used for non-Hodgkins lymphoma.

Antibody-directed enzyme prodrug therapy (ADEPT) involves the application of cancer

associated monoclonal antibodies which are linked to a drug-activating enzyme. Subsequent

systemic administration of a non-toxic agent results in its conversion to a toxic drug, and

resulting in a cytotoxic effect which can be targeted at malignant cells. The clinical success of





ADEPT treatments has been limited to date [16]. However it holds great promise, and recent

reports suggest that it will have a role in future oncological treatment.

Immunoliposomes are antibody-conjugated liposomes. Liposomes can carry drugs or

therapeutic nucleotides and when conjugated with monoclonal antibodies, may be directed

against malignant cells. Although this technique is still in its infancy, significant advances have

been made. Immunoliposomes have been successfully used in vivo to achieve targeted delivery

of tumour-suppressing genes into tumours, using an antibody fragment against the human

transferrin receptor. Tissue-specific gene delivery using immunoliposomes has also been

achieved in brain, and breast cancer tissue [17].

Fig. 2: Monoclonal antibodies for cancer. ADEPT, antibody directed enzyme prodrug

therapy; ADCC, antibody dependent cell-mediated cytotoxicity; CDC, complement

dependent cytotoxicity; MAb, monoclonal antibody; scFv, single-chain Fv fragment [18].





Autoimmune diseases

Monoclonal antibodies used for autoimmune diseases include infliximab and adalimumab, which

are effective in rheumatoid arthritis, Crohn's disease and ulcerative Colitis by their ability to bind

to and inhibit TNF-α [19]. Basiliximab and daclizumab inhibit IL-2 on activated T cells and

thereby help preventing acute rejection of kidney transplants [19]. Omalizumab inhibits human

immunoglobulin E (IgE) and is useful in moderate-to-severe allergic asthma.

Conclusion

The limitation in the production of monoclonal antibody by hybridoma technology opened doors

for improvements. This advancement in hybridoma technology led to an appreciable increase in

the quality and quantity of monoclonal antibody produced hybridoma technology. These

monoclonal antibodies are essential tools in biochemical research, Diagnosis,

immunopurification and immunotherapy. With recent outbreak of Ebola virus infection, much

research should be carried out towards the efficacious treatment of this infection using

monoclonal antibody which will further highlight the so-called Monoclonal Antibody Therapy.

References

1. Bretton, PR, Melamed, MR, Fair, WR, Cote, RJ (1994). Detection of occult micrometastases

in the bone marrow of patients with prostate carcinoma. Prostate. 25(2), 108-14.

2. http://en.wikipedia.org/wiki/Hybridoma_technology #Method

3. Franklin, WA, Shpall, EJ, Archer, P, Johnston, CS, Garza-Williams, S, Hami, L, Bitter MA,

Bast RC, Jones, RB (1996). Immunocytochemical detection of breast cancer cells in marrow

and peripheral blood of patients undergoing high dose chemotherapy with autologous stem

cell support. Breast Cancer Res Treat. 41(1), 1-13.

4. Ghosh, AK, Spriggs, Al, Taylor-papadimitriou, J and Mason, DY (1983).

Immunocytochemical staining of cells in pleural and peritoneal effusions with a panel of

monoclonal antibodies. J Clin Pathol, 36, 11541164.

5. Kvalheim, G (1996). Detection of occult tumour cells in bone marrow and blood in breast

cancer patients—methods and clinical significance. Acta Oncol, 35, 13-8.





6. http://www.molecular-plant-biotechnology.info/hybri doma-and-monoclonal-antibodies-mabs/

raising-theantibodies.htm

7. Elements of biotechnology, P.K. GUPTA (1st EDITION): 2006-2007, (203-211).

8. ^ "immunotherapies definition”. Dictionary.com. Retrieved 2009-06-02

9. http://en.m.wikipedia.org/wiki/Monoclonal_antibody_therapy

10. Prof FC Breedveld (2000). "Therapeutic monoclonal antibodies" Lancet. doi

:10.1016/S0140-6736(00)01034-5 .

11. Köhler G, Milstein C (August 1975). "Continuous cultures of fused cells secreting antibody of

predefined specificity". Nature 256 (5517): 495–7. Bibcode: 1975 Natur.256..495K. doi:

10.1038/256495a0. PMID 1172191.

12. Nadler LM, Stashenko P, Hardy R, et al. (September 1980). "Serotherapy of a patient with a

monoclonal antibody directed against a human lymphoma-associated antigen". Cancer Res.

40 (9): 3147–54. PMID 7427932.

13. Ritz J, Schlossman SF (January 1982). "Utilization of monoclonal antibodies in the treatment

of leukemia and lymphoma". Blood 59 (1): 1–11. PMID 7032624.

14. a b c Stern M, Herrmann R (April 2005). "Overview of monoclonal antibodies in cancer

therapy: present and promise". Crit. Rev. Oncol. Hematol. 54 (1): 11–29. doi

:10.1016/j.critrevonc.2004.10.011 . PMID 15780905.

15. a b Hudson PJ, Souriau C (January 2003). "Engineered antibodies". Nat. Med. 9 (1): 129–

34. doi: 10.1038/nm0103-129 .PMID 12514726.

16. Francis RJ, Sharma SK, Springer C, et al. (2002). "A phase I trialof antibody directed

enzyme prodrug therapy (ADEPT) in patientswith advanced colorectal carcinoma or other

CEA producingtumours. Br J Cancer 87 (6): 600–7. doi :10.1038/sj.bjc.6600517 .PMC

2364249 . PMID 12237768.

17. Krauss WC, Park JW, Kirpotin DB, Hong K, Benz CC (2000). "Emerging antibody-based

HER2 (ErbB-2/neu) therapeutics". BreastDis 11: 113–124. PMID 15687597.

18. Modified from Carter P (November 2001). "Improving the efficacy of antibody-based cancer

therapies". Nat. Rev. Cancer 1 (2): 118–29. doi : 10.1038/35101072 . PMID 11905803.

19. a b Rang, H. P. (2003). Pharmacology. Edinburgh: Churchill Livingstone. p. 241. ISBN 0-

443-07145-4.





Lyophilisation: A Method of Preserving

Biologicals

Dr Sakshi Bhadouriya, M.V.Sc (Vet. Virology, IVRI, Bareilly)

Dr Sunil Singh Tomar, M.V.Sc (Vet. Clinical Medicine)

Introduction

Lyophilisation (also known as Freeze Drying or Cryodesiccation) is a dehydration process

typically used to preserve a perishable material or make the material more convenient for

transport. With proper packaging and storage, freeze-dried products can be stored for a very

long time without any appreciable loss of quality even at room temperature. Freeze-drying

works by freezing the material and then reducing the surrounding pressure and adding enough

heat to allow the frozen water in the material to sublime directly from the solid phase to the gas

phase. Lyophilisation technology is used to freeze-dry products such as biologicals, bacterial

cultures, analytical chemistry moieties, and therapeutic molecules (e.g., antibodies, vaccines,

drugs, and heat-sensitive proteins). Lyophilizing such products, particularly liquid formulations,

vastly increases their shelf-life and stability. Freeze-dried products have an optimal capacity to

take up water again (reconstitution, rehydration).

The Principles of Freeze-Drying

At atmospheric pressure (approx. 1,000 mbar) water can have three physical states:

1. Solid;

2. Liquid;

3. Gaseous.

Below the triple-point (for pure water at 0°C), only the solid and the gaseous states exists. The

principle of freeze/sublimation-drying is based on this physical fact. The ice in the product is

directly converted into water vapour without passing through the “fluid state”.





Process of Freeze Drying

The process consists of three separate, unique, and interdependent processes:

Freezing

Primary drying (sublimation),

Secondary drying (desorption).

In a lab, this is often done by placing the material in a freeze-drying flask and rotating the flask

in a bath, called a shell freezer, which is cooled by mechanical refrigeration, dry ice and

methanol, or liquid nitrogen. On a larger scale, freezing is usually done using a freeze-drying

machine. In this step, it is important to cool the material below its triple point, the lowest

temperature at which the solid and liquid phases of the material can coexist. This ensures that

sublimation rather than melting will occur in the following steps. Larger crystals are easier to

freeze-dry. To produce larger crystals, the product should be frozen slowly or can be cycled up

and down in temperature. This cycling process is called annealing. However, in the case of





food, or objects with formerly-living cells, large ice crystals will break the cell walls (a problem

discovered, and solved, by Clarence Birdseye), resulting in cell destruction, and, in the case of

rehydrated foods, a poor texture. In this case, the freezing is done rapidly, in order to lower the

material to below its eutectic point quickly, thus avoiding the formation of ice crystals. Usually,

the freezing temperatures are between −50 °C and −80 °C. The freezing phase is the most

critical in the whole freeze-drying process, because the product can be spoiled if badly done.

Amorphous materials do not have a eutectic point, but they do have a critical point, below which

the product must be maintained to prevent melt-back or collapse during primary and secondary

drying.

Primary Drying

During the primary drying phase, the pressure is lowered (to the range of a few millibars), and

enough heat is supplied to the material for the water to sublimate. The amount of heat

necessary can be calculated using the sublimating molecules’ latent heat of sublimation. In this

initial drying phase, about 95% of the water in the material is sublimated. This phase may be

slow (can be several days in the industry), because, if too much heat is added, the material’s

structure could be altered. In this phase, pressure is controlled through the application of partial

vacuum. The vacuum speeds sublimation, making it useful as a deliberate drying process.

Furthermore, a cold condenser chamber and/or condenser plates provide a surface(s) for the

water vapour to re-solidify on. This condenser plays no role in keeping the material frozen;

rather, it prevents water vapor from reaching the vacuum pump, which could degrade the

pump's performance. Condenser temperatures are typically below −50 °C (−60 °F). It is

important to note that, in this range of pressure, the heat is brought mainly by conduction or

radiation; the convection effect is considered to be inefficient.

Secondary drying

The secondary drying phase aims to remove unfrozen water molecules, since the ice was

removed in the primary drying phase. This part of the freeze-drying process is governed by the

material’s adsorption isotherms. In this phase, the temperature is raised higher than in the

primary drying phase, and can even be above 0 °C, to break any physico-chemical interactions





that have formed between the water molecules and the frozen material. Usually the pressure is

also lowered in this stage to encourage desorption (typically in the range of microbars, or

fractions of a pascal). However, there are products that benefit from increased pressure as well.

After the freeze-drying process is complete, the vaccum is usually broken with an inert gas,

such as nitrogen, before the material is sealed. At the end of the operation, the final residual

water content in the product is extremely low, around 1-4%.

Flow Chart of Lyophilisation

Material in vial in shelves is pre-chilled the shelves to -45 c & initiate the drying when material

temp -40 c.

Vaccine harvest + stabiliser

Product in direct contact vd shelves (-40 c)

Condenser temp (-55 to -60 c)

Then primary drying starts (1% to 4%)

Remove crystalline ice by sublimation when water sublimes it takes energy so product is cooled

Energy of sublimated water is release to condenser so condenser tends to warm

Self heating & refrigeration & compressor works counteractly.

Product have residual 5-10 % moisture (not frozen so all ice is removed in this)

Key Component of Freeze Drying

Vaccum chamber

Lyophilisation shelves





Heat transfer fluid

Product

Condenser

A mechanical or cryogenic refrigeration system

Heater

Vaccum pump

Control hardware and software

Freeze Drying Methods

Three methods of Freeze Drying are commonly used:

1. Manifold drying

2. Batch drying

3. Bulk Drying

Properties of freeze-dried products

If a freeze-dried substance is sealed to prevent the reabsorption of moisture, the substance may

be stored at room temperature without refrigeration, and be protected against spoilage for many

years. Preservation is possible because the greatly reduced water content inhibits the action of

microorganisms and enzymes that would normally spoil or degrade the substance. Freeze-

drying also causes less damage to the substance than other dehydration methods using higher

temperatures. Freeze-drying does not usually cause shrinkage or toughening of the material

being dried. Freeze-dried products can be rehydrated (reconstituted) much more quickly and

easily because the process leaves microscopic pores. The pores are created by the ice crystals

that sublimate, leaving gaps or pores in their place. This is especially important when it comes

to pharmaceutical uses. Freeze-drying can also be used to increase the shelf life of some

pharmaceuticals for many years

Disadvantages of Freeze Drying

Maintaining frozen storage is costly and takes up a lot of space

Transportation of frozen materials can be difficult and expensive





Failure of freezing equipment would risk the total loss of the product

Conventional drying methods also have a major disadvantage as the high temperatures

used can cause chemical or physical changes to the product.

References

1. LYOPHILIZATION: Univ.Prof. Eng. Dumitru MNERIE, PhD “POLITEHNICA” University

of Timisoara ROMANIA

2. T. A. Jennings, “Lyophilization - Introduction and Basic Principles”, Interpharm Press,

Buffalo Grove, IL 1999.

3. A. I. Liapis and R. Bruttini, in A.S. Mujumdar ed., “Handbook of industrial Drying” vol.1,

2nd ed., Marcel Dekker, New York and Basel, 1995, 309 – 343.





Rapid diagnosis of acute respiratory infections

by multiplex endpoint PCR technology 1Aurelian Udristioiu, 2Manole Cojocaru, 3Dana Alexandra Maria Panait, 4Nica

Badea Delia

1Clinical Laboratory, Department of Hematology, Emergency County Hospital Targu Jiu

& UCB University, Romania, E-mail: [email protected]

2Titu Maiorescu University, Faculty of Medicine, Physiology Department, Bucharest,

Romania; E-mail: [email protected]

3Titu Maiorescu University, Faculty of Medicine, Microbiology Department, Bucharest,

Romania, E-mail [email protected]

4Constantin Brancusi University, Faculty of Medical Science and Behavioral, Targu Jiu,

Romania, Email: [email protected]

Introduction

The multiplex endpoint PCR technology offers a number of potential advantages, results are

available in a matter of hours rather than days, the extreme sensibility facilitates detection of

even minutes the amounts of pathogen DNA in clinical samples and the test is not significantly

affected by prior administration of antibiotics.

Aim

The aim of this work was to rapidly identify the antibiotic resistance the monitoring of pathogen

growth at the patients admitted in Hospitalization Intensive Care Unit of Emergency County

Hospital Targu Jiu with the diagnosis of Community Acquired Pneumonia, (CAP), in months

December/2013-March/2014.

Method

The Analyzer Unyvero™ Pneumonia Application was used in detection of pneumonia

associated pathogens and their antibiotic resistance genes using the Pneumonia Unyvero™

System, following PCR pathogen species with sequencing of the amplified microbial DNA.





Results

The main pathogens of community acquired pneumonia from the cohort study, 36 cases, (20

males in mean age 35-66 years and 16 females in mean age 40-55 mean years) were

Streptococcus pneumonia (16 cases), Staphilococcus aureus, (10 cases), Klebsiella

pneumonia (5 cases), and other important agents were “atypical”, such as Haemophilus

Influenzae, Chlamidophila pneumonie and Moraxela cataralis.

A case with Acinetobacter baumani and Proteus Sp. was also widely resistance to mefA gene /

ermB gene as all cases of analyzed. The more frequency of genes resistant (29 cases) are

ermA gene / ermC / ermB for Staphilococcus aureus and the gene tem+shv / gene / ctx-M with

the chromosomal mutation (7 cases), as gyrA83_87 Ecoli / Pseu for Klebsiella pneumonia

agents. Also most resistance antibiotics were Makrolides, (29 cases and Lincosamides (6

cases) and this cases have had the chromosomial integrates. The most resistance microbe,

Pseudomonas aeruginosa (1 case), has been registered as multi drugs resistance [MDR]*.

Fig. 1: Microorganism results in Pneumonia Panel





No.

IDL

Microorganisms

detected

Antibiotic resistance Gene resistance

1310_1

Klebsiella pneumonia

Makrolides /],

[ermB],].Lincosamides

ermB gene

1310_2 Streptoccocus Sp.

Makrolides. ermB gene/tem gene

1310_3

Staphilococcus

aureus

Other/Fungi:

Chlamidophila

pneumonie

Penicilins (tem) ermB gene/tem gene

Chromosomal mutation;

Pseud. aeruginosa, (gyrA83-

Ecoli

1310_4 Klebsiella pneumonia

Makrolides / Lincosamides ermB gene

1310_5

Proteus Sp.

Other/Fungi:

Haemophilus

Influenzae,

Chlamidophila

pneumonie

Makrolides

Oxacilin

ermB gene/oxa51


Escherichia Coli (gyrA83-

87_Ecoli ).

1311_1

Streptococcus

pneumonia Moraxela

cataralis

Makrolides,

Oxacillin Lincosamides,

ermB gene/tem gene/ mecA gene

1311_2

Streptococcus

pneumonia

Makrolides / [mefA],


Penicilins (tem)

mefA gene / ermB gene / tem

1311_3

Streptococcus

pneumonia

Pseudomonas

aeruginosa



[MDR]* [int1].

mefA gene / ermB gene

[int1gene].

1311_4 Klebsiella pneumonia Penicilins (shv) mefA gene / shv gene

[int1gene] /sul 1 gene


(gyrA83_3Pseu).





[MDR]*-Multi drugs resistance

Table 1. Resistance markers of the Pneumonia panel and the resulting antibiotic

resistances

Photo 1. Analyzer Unyvero™ Pneumonia

Conclusion

The Unyvero™ results have been available 2 days before the primary microbiology report and 3

days before the final confirmation results, obtained by microbiology culture. The Unyvero

Analyzer only provides rapid data to support the therapeutic decision of currant medic.

1311_5 Streptococcus

pneumonia

Acinetobacter

baumani



mefA gene / ermB gene

1312_1

Staphilococcus

aureus

Klebsiella pneumonia



3rd Gen Cephalosporins

[tem+shv], [ctx-M].

ermA gene / ermC / ermB

tem+shv / gene / ctx-M g


gyrA83_87 Ecoli / Pseu





Bioweapons: A new trend in emerging Sciences 1Hasnain Nangyal, 2Noor Nasir Khattak, 3Sikander Khan Sherwani

1Department of Botany, Hazara University, Khyber Pakhtoonkhwa, Pakistan

2Department of Gynocology and Obteristics, Kohat Medical College, Khyber

Pakhtoonkhwa, Pakistan

3Department of Microbiology, FUUAST, Karachi, Pakistan

Corresponding Author Email: [email protected]

Basically different biologists defined this term in different ways:

1. Bioweapons are the biological agents derived from the living organisms that are capable

of infecting and causing both sickness and death in people, animals and plants.

2. Bioweapons sometime called biowarfare which means that intentional use of

microorganisms or toxins derived from living organisms of as an act of war or political

violence with intent to cause death or disease in human, animal or in plants (1)

3. Biological warfare (BW)—also called germ warfare. It is the use of biological toxins or

infectious agents such as bacteria, viruses, and fungi with intent to kill or incapacitate

humans, animals or plants as an act of war.

4. Biological weapons (often termed "bio-weapons", "biological threat agents", or "bio-

agents") are living organisms or replicating entities (viruses, which are not universally

considered "alive") that reproduce or replicate within their host victims.

5. Biological warfare agents are likely to be selected for their ability to either incapitate or

kill human targets of attack.

Types of Bioweapons:

There are seven types of biological agents:

1: Parasites

2: Fungi and yeasts

3: Bacteria

4: Rickettsia and chlamydia





5: viruses

6: Prions

7: Toxins

8: Insects

Among the biological agent only bacteria, viruses and toxins are considered when referring to

agents that can be used in a biological attack. While toxins are included in the list of biological

agents, they are not living organisms, but small proteins produced by bacteria that can poison to

a person, animal or plant. Bacteria, viruses and toxins can be spread through the contamination

of food, water or fomites; via vectors such as insects; or as aerosols suspended in wet or dry

formulations (2).

Entomological (insect) warfare is also considered a type of biological weapon. This type of

warfare is distinct from nuclear warfare and chemical warfare. None of these falls under the

term conventional weapons which are primarily effective due to their destructive potential.

Biological weapons (BWs) deliver toxins and microorganisms, such as viruses and bacteria, so

as to deliberately inflict disease among people, animals, and agriculture. Biological attacks can

result in destruction of crops, temporarily discomforting a small community, killing large numbers

of people, or other outcomes.

Possible Biological Warfare

Agents

Bacteria (examples)

Viruses (examples)

Toxins (examples)

Bacillus anthracis (anthrax)

Variola virus (smallpox)

Staphylococcal

enterotoxin B

Yersinia pestis (plague)

Equine encephalitis viruses (viral

equine

encephalitides)

Ricin





Francisella tularensis

(tularemia)

Arenaviruses,bunyaviruses,

filoviruses

Botulinum toxins

Brucella species (brucellosis)

flaviviruses (hemorrhagic fevers)

Trichothecene mycotoxins

Coxiella burnetii (Q fever)

- Saxitoxin

Classification of Bioweapons:

There are four classifications for how biological agents can be used to harm or kill a person,

animal or plant.

1: Biological Warfare (BW): is the military use of biological agents, where targets of agents are

predominately soldiers, governments, or resources that might hinder a nation’s ability to attack

and/or defend it.

2: Bioterrorism (BT): is the threat or use of biological agents that, like most forms of terrorism,

is intended to make political, religious or personal statements to governments and populations

through attacks primarily aimed at civilians or resources that affect the civilian economy. With

few exceptions, bioterrorism is non-state sponsored.

3: Biocrime (BC): is the threat or use of biological agents for individual objectives such as

revenge or financial gain.

4: Bioaccident (BA): It is defined as the unintentional release of an agent from a laboratory or

other facility. Biocrimes and Bioaccidents comprise events that typically have small effects on

populations and do not require specific plans for large-scale preparedness and response (3).

Categories of Bioweapons:

The Critical Agent List classifies a relatively short list of possible biological weapons to be used

in either biological warfare or bioterrorism, and was created by the Centers for Disease Control

and Prevention (CDC) in conjunction with military, intelligence, medical and public health

agencies: (20)

Category A includes the highest priority agents because they are most likely to cause mass

casualties, create panic and require a specific public health response. According to intelligence





sources, these are the agents that are most likely to be used in a future attack, and are being

researched and weaponized by biowarfare programs around the world.

Category B is the second highest priority agents, including those agents that could contaminate

food or water, are relatively easy to disseminate, and require enhanced disease surveillance

and diagnostic capacity. Many of these agents, such as brucellosis, glanders and ricin, were

either weaponized by state-sponsored programs in the past, or utilized successfully in biological

warfare or terrorist incidents

Category C includes emerging pathogens that could be weaponized in the future because of

the relative ease of accessing, producing and disseminating the agents, as well as the high

levels of morbidity and mortality these agents would cause.(4)

Although the CDC has been able to categorize a list of agents that are likely to be used as

bioweapons, there are additional diseases and variations of biological agents that are of great

concern. The Critical Agent List specifies only twenty-one diseases, but there are at least

seventy different types of biological agents that can be weaponized, not including agents that do

not already exist in nature. Of the disease caused by these seventy agents, only 20-30% is

currently treatable through reliable methods.(5)

Critical list of Biological agents

Category A Category B Category C

Small pox Q-fever nipah virus

Anthrax Brucellosis Hantavirus

Plague Glanders multidrug-resistant TB

Tularemia melioidosis

hemorrhagic fever viral encephalitis

Ebola Ricin

(21)

History

There is a long history of nations and peoples using biological agents as weapons. Many

examples of use have been cited from as long ago as 190 BC, where Hannibal used venomous

snakes to disrupt the enemy ships of Pergamus in Eurymedon. Biological weapons have a long





history of use. In 1346, the invading Tartar army catapulted the bodies of plague victims into the

Crimean Peninsula city of Kaffa and infected its citizens. Another example of biological warfare

often referred to in the historical record is the use of smallpox during the French and Indian War

in 1763. Through the combined ingenuity of British Officers Sir Jeffrey Amherst and Colonel

Henry Bouquet, smallpox infested blankets were given to the Indians at Fort Pitt, setting off an

epidemic of smallpox that rendered the Indian tribes incapable of fighting off the British settlers

(6). In this instance, it is not clear if the blankets themselves caused the outbreak of smallpox or

whether it was due to previous exposure to the Europeans. Regardless, this method of

biological warfare was used again during the American Civil War, when Dr. Luke Blackburn of

Kentucky sold smallpox and yellow fever infected clothing to Union troops in an effort to support

the efforts of the Confederacy. There were also reports that the Germans used plague against

the Russians in 1915, and attempted to use cholera against Italy (7). Between 1933 and the

late 1940’s, Japan researched biological warfare at a compound known as Unit 731, where over

10,000 prisoners of war used for research purposes died of anthrax, meningitis, cholera and

plague. Japan followed its experiments with prisoners of war by using biological agents against

the people of China, causing outbreaks of typhus, cholera and plague, resulting in the deaths of

tens of thousands of Chinese civilians.(19) One method used by the Japanese in Manchuria

was dropping rice and plague infected fleas out of airplanes. The rice attracted rats, which then

became infected with the fleas, thus creating efficient epidemiologic conditions by which the

disease spread to humans over a large geographic area(8). The 2001 anthrax letter attacks in

the United States infected 22 people and killed five (Biological weapon technology, 1998).The

Genoese forces retreated back to Genoa, Venice and other Mediterranean ports, sparking the

second wave of the plague epidemic in Europe. (9)

Characterizing the Threat of Biological Weapons

On 11 September 2001, terrorists used hijacked passenger planes loaded with jet fuel as

explosion devices at the Pentagon and World Trade Center, killing approximately 3000. This

attack awakened the American public to the idea that terrorists have the will and the means of

inflicting harm on citizens of the United States within their national borders. Following the

September 11th attack, letters with anthrax were mailed to a targeted list of media and political





personalities.(18) There are many reasons why biological agents are effective means of

spreading terror which include followings;

Availability

Biological agents are often simpler to acquire and produce than nuclear, chemical or some

conventional weapons. The material for biological agents can be easily grown or purchased.

Some agents, such as anthrax or brucellosis, occur naturally in animals in certain parts of the

world, and individuals can easily travel the globe to acquire biological agents from regions

where such diseases occur naturally. For example, the Aum Shinrikyo cult was reported to

have gone to Zaire to collect strains of Ebola for use in its bioweapons program. Until recently,

anyone could order agents from supply houses around the world (10).

Access to Technology

Technological advances over the last two decades in the field of biology have made it easier to

produce biological weapons. Techniques once considered beyond the capacity of scientists are

now commonplace, particularly in the field of genetic engineering. Bioengineers are now armed

with the knowledge needed to create antibiotic resistant strains of agents, combine aspects of

two agents, weaponize agents, and possibly target particular populations. The creation of

antibiotic resistant strains of anthrax was a key component of the Soviet biowarfare program28.

The Soviets were also successful in creating more virulent strains of the disease, capable of

bypassing the available anthrax vaccine currently given to United States active duty forces29.

Bioengineers are also now able to combine two agents to complicate the presentation of a

disease in humans. Again, it was the Soviets who first used this technology by using

recombinant DNA to combine features of smallpox and Ebola, and in 1995, Russian scientists

presenting at a scientific conference in England, reported that they had combined a food

poisoning organism with anthrax (11).

Access to Information

Information on how to create sophisticated biological weapons can be taken from articles

published in the scientific literature on a variety of topics, but it requires a trained scientist to





understand the relevance of published findings to the creation of weapons. However,

information on how to weaponize simpler biological agents is widely available and

understandable to people with limited technological training.

Lethality

Biological agents can be extremely lethal, with some agents creating much more deadly affects

than others. According to the Department of Defense, ten kilograms of anthrax could cause

more casualties that a 10- kiloton nuclear weapon (12).

Lack of Collateral Damage

While biological agents may indiscriminately kill or injure humans, animals and plants, they do

not destroy structures. This aspect of biological agents may be of interest to groups who desire

to kill residents of an area, but protect surrounding assets, such as religious shrines, historical

buildings, or industrial plants (13).

Plague as a past bioweapon:

Plague is a zoonotic disease caused by the bacterium Yersinia pestis, typically harbored by wild

rodents (Fig). The plague epidemic that swept through Europe, the Near East, and North Africa

in the mid-14th century was probably the public health disaster in recorded history. An

estimated one-quarter to one-third of Europe’s population died from plague during the 14th

century pandemic, and North Africa, the Near East, and perhaps the Far East had similar high

levels of mortality.(14) However, the first recorded plague andemic began in 541 in Egypt when

the world population as considerably smaller and decimated an even greater ercentage of

the population.

Advantages and Disadvantages of Biological Weapons

Advantages

1: The potential deadly or incapacitating effects on a susceptible population

2: The self-replicating capacity of some biological agents to continue proliferating in the affected

individual and potentially in the local population and surroundings





3: The relatively low cost of producing many biological weapons

4: The insidious symptoms that can mimic endemic diseases

5: The difficulty of immediately detecting the use of a biological agent, owing to the current

limitations in fielding a multi-agent sensor system on the battlefield, as well as to the prolonged

incubation period preceding onset of illness (or the slow onset of symptoms) with some

biological agents(15)

6: The sparing of property and physical surroundings (compared with conventional or nuclear

weapons)





Disadvantages

1: The danger that biological agents can also affect the health of the aggressor forces

2: The dependence on prevailing winds and other weather conditions on effective dispersion

3: The effects of temperature, sunlight, and desiccation on the survivability of some infectious

organisms

4: The environmental persistence of some agents, such as spore-forming anthrax bacteria,

which can make an area uninhabitable for long periods

5: The possibility that secondary aerosols of the agent will be generated as the aggressor

moves through an area already attacked

6: The unpredictability of morbidity secondary to a biological attack, since casualties (including

civilians) will be related to the quantity and the manner of exposure

7: The relatively long incubation period for many agents, a factor that may limit their tactical

usefulness

8: The public’s aversion to the use of biological warfare agents (16)

Indications of Possible Biowarfare Attack

• A disease entity that is unusual or that does not occur naturally in a given geographic area, or

combinations of unusual disease entities in the same patient populations

• Multiple disease entities in the same patients, indicating that mixed agents have been used in

the attack

• Large numbers of both military and civilian casualties when such populations inhabit the same

area

• Data suggesting a massive point-source outbreak

• Apparent aerosol route of infection

• High morbidity and mortality relative to the number of personnel at risk

• Illness limited to fairly localized or circumscribed geographical areas

• Low attack rates in personnel who work in areas with filtered air supplies or closed ventilation

systems

• Sentinel dead animals of multiple species





• Absence of a competent natural vector in the area of outbreak (for a b iological agent that is

vector-borne in nature) (17).

References:

1. Manchee R, Stewart W.1988. The decontamination of Gruinard Island. Chem Br.690–

691.

2. Nally R. E, Morrison MB, Stark M. 1993. Effectiveness of medical defense interventions

against predicted battle field levels of Bacillus anthracis. Science Applications

International.32-35

3. Patrick W.C.1992. Overview of biological warfare. Frederick, Unpublished

manuscript.11-14

4. North Atlantic Treaty Organization. NATO Handbook on the Medical Aspects of NBC

Defensive Operations. A-Med-P6, part 2, Biological. June 1992.

5. World Health Organization. Health Aspects of Chemical and Biological Weapons: Report

of a WHO Group of Consult- ants . Geneva, Switzerland: WHO; 1970.

6. Huxsoll D, Patrick W.C, Parrott C.1987. Veterinary services in biological disasters.

Journal of veterinary sciences. 714–722.

7. Department of the Army. Special Report to Congress. US Army Activities in the US

Biological Defense Programs,1942–1977. Vols 1 and 2. Washington, DC: DA. 24 Feb

1977. Unclassified.

8. Special Subcommittee on the National Science Foundation of the Committee on Labor

and Public Welfare, US.Senate. Chemical and Biological Weapons: Some Possible

Approaches for Lessening the Threat and Danger. Washing-ton, DC: US Government

Printing Office; May 1969.

9. Special Subcommittee on the National Science Foundation, Committee on Labor and

Public Welfare. Chemical and Biological Weapons. Washington, DC: US Government

Printing Office; May 1969.

10. Ursano R. Combat stress in the chemical and biological warfare environment. Aviation

Space & EnvironmentalMedicine. 1988;59(12):1123–1124.





11. Mojecki, J.A.1994. Mission-essential training and weapons of mass destruction. Military

Review. 91–93.

12. Geissler E, ed.1986. Biological and Toxin Weapons Today. Stockholm International

Peace Research Institute. Oxford, England: Oxford University Press

13. Douglas, J.D. Livingstone, N.C.1987. America the Vulnerable: The Threat of Chemical

and Biological Warfare. Lexington,Mass: Lexington Books.

14. US Department of State. International Terrorism. Washington, DC: DOS; 1989. GIST.

15. Zilinskas, R.A.1990. Terrorism and biological weapons: Inevitable alliance? Perspect

Biol Med. 44–72.

16. Bates S.1992. Fairfax man accused of anthrax threat. Washington Post.

17. Kupperman, R.H, Smith, D. M.1991. Coping with biological terrorism. Washington, DC..

18. US Army Medical Research Institute of Infectious Diseases. Medical Management of

Biological Casualties Hand- book . Fort Detrick, Frederick, Md: USAMRIID; August 1993.

19. Wiener, S.L, Barrett, J.1986. Biological warfare defense. In trauma Management for

Civilian and Military Physicians. Philadelphia, Pa: WB Saunders.507–528.

20. Caudle L.1994. Major, Medical Corps, US Army. Officer in Charge, Problem Definition

and Assessment Team, Operation Vigilant Warrior. Personal communication.

21. Khan A, et al. Public health preparedness for biological terrorism in the USA. Lancet

2000; 356:1179





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