eChapters

In animals with a long gestation period, such as domestic animals, the acquired immune system is fully developed at birth, but cannot function at full adult levels for several weeks. The complete development of the acquired immunity depends on antigenic stimulation. The proper development of B cells and B cell antigen receptor (BCR) diversity requires clonal selection and antigen driven cell multiplication. Thus, the newborn animals are vulnerable to infection for the first few weeks of life. Hence, the temporary assistance in defending themselves at this time is provided by the mother in the form of antibodies and possibly T cells.
 
Development of the Immune System

The thymus is first lymphoid organ to develop, followed closely by the secondary lymphoid organs. B cells appear soon after the development of spleen and  lymph nodes, but antibodies are not usually found until late in foetal life, if at all. The ability of fetus to respond to antigens develops very rapidly after the lymphoid organs appear, but all antigens are not equally capable of stimulating fetal lymphoid tissue. The immune system in a series of steps, each step permitting the foetus to respond to more antigens. These steps are driven by a gradual increase in the use of gene conversion or somatic mutation to antibody diversity. The ability to mount cell mediated immune (CMI) responses develops at the time as antibody production. Data from humans suggest that T cell receptor diversity is limited in the fetus and neonate and that cytokine production may be low. This may simply be due to their lack of to foreign antigens.

A. Answer the following

Q.1.    What is the difference between apoptosis and necrosis?

Ans.    In apoptosis, there is pronounced decrease in cell volume, modification of cytoskeleton, condensation of chromatin and degradation of DNA into fragments. Due to these changes, an apoptotic cell sheds tiny membrane bound apoptotic bodies containing intact organelles. Macrophages phagocytose apoptotic bodies and cells in the advanced stages of apoptosis. But there is no release of proteolytic and the lytic enzyme, cationic proteins and oxidizing molecule in the surrounding tissue and no local inflammatory response. On the other hand, in necrosis cells swell and burst releasing their contents and possibly triggering a damaging inflammatory response.

A. Answer the following.

Q.1.     What are the different phagocytic cells ?
Ans.     The phagocytic cells of mammals belong to two complementary systems (1) The myeloid system consists of cells that are rapidly phagocytic but incapable of sustained effect. (2) Mononuclear phagocytic system act more slowly but capable of repeated phagocytosis.

Q.2.     How 3 types of granulocytes are differentiated ?
Ans.    Three types of granulocytes are distinguished by the staining property of their granules. Cells whose granules take up basic dyes such as haematoxylin (a blue dye) are called basophils, whose granules take up acidic dyes such as eosin (a red dye) are called eosinophils and those that take up neither basic nor acidic dyes are called neutrophils.

Feed accounts for a significant cost of production in livestock and poultry. In case of poultry, it represents 65% cost of the production in broilers and 75% in layers. Presently the live stock industry faces a huge challenge due to the high cost of feed ingredients and shortage of the conventional feed ingredients. Over the years, efforts have been made to use unconventional feed ingredients. However, such feed ingredients have many anti nutritional factors that adversely affect the performance of livestock and poultry. Due to the presence of these incriminating factors, the unconventional feed ingredients are poorly digested in livestock and poultry. These poorly digested feed ingredients results in bacterial overgrowth, which becomes a potential substrate for bacterial fermentation leading to intestinal disorders and disease. Application of antibiotics and other anti-microbials help to overcome the ill effects. However, the ban on the use of sub therapeutic level of antibiotics in many countries has increased the relevance of use of exogenous enzymes in the feed of livestock and poultry. These exogenous feed enzymes improve the nutritive value of different feed stuffs, economize livestock and poultry rearing and reduce environmental pollution.

Molecular diagnostics has witnessed groundbreaking technological advances in recent times, starting from automated sample preparation to real time amplification technology. Possibility to develop and run assays for ever increasing number of clinical queries has become effortless due to the technological advances. However, the state of the art in quality assurance and quality control practices for molecular diagnostics has seldom been up to the expectations if one considers accuracy for once in a lifetime genetic tests or new genetic test targets appearing almost every single day. In the face of such issues, clinical laboratories are struggling to develop appropriate quality assurance programs for the molecular diagnostic tests they conduct. In addition, legislation and government policies threaten to increase oversight of genetic tests to a level not seen before in laboratory medicine, for example, FDA’s guidance on in vitro diagnostic multivariate index assays (IVDMIA).

To relate patient results to previous test results or to absolute values in clinical practice guidelines, those results need to be comparable across time and methods. This may either be achieved by designing the identical value across methods and test versions or by using reliable and stable conversions. Therefore, the establishment of international standards and reference materials is of paramount importance. Monitoring molecular test system outputs and statistical analysis might provide a good way to obtain such standards; however, such traditional QC (Quality Control) strategies can be slow to take root in molecular diagnostics.This chapter highlights general and specific issues relevant for quality assurance and quality control in the routine molecular diagnostics laboratory and guidelines for designing quality manuals for a molecular diagnostic laboratory.

A. Answer the following.

Q.1.     What are tumour specific transplantation antigens (TSTAs) or tumour specific antigens (TSAs) ?
Ans.     Some antigens present on the surface of malignant cells are unique to the cancerous cells and are not present on their normal counterparts. They are called tumour specific transplantation antigens (TSTAs) or tumour specific antigens (TSAs).

Q.2.     What are tumour associated antigens (TAAs) ?
Ans.    Some antigens present on the tumour cells that are not different qualitatively from those found on normal cells but are overexpressed and present in increased number on the cancer cells as products of cellular oncogenes. They are called tumour associated antigens (TAAs). Ex the high level of a growth factor receptor due

This chapter describes the cells, tissues and organs responsible for immune defence. It examines lymphocytes, macrophages, dendritic cells, neutrophils, mast cells, natural killer cells and plasma cells, emphasizing their respective functions. Primary and secondary lymphoid organs—including bone marrow, thymus, bursa of Fabricius, spleen, lymph nodes and mucosa-associated lymphoid tissue—are discussed. The chapter also covers lymphocyte maturation, antigen presentation, phagocytosis, immune-cell surface markers and the organization of lymph-node regions. Positive and negative selection of T lymphocytes and the formation of memory cells are included. Together, these topics explain how immune components develop, communicate and coordinate effective responses against infectious agents.

Viruses can be cultivated only in living cells. Laboratory animals, embryonated avian eggs and cell cultures are three systems available for this purpose. These three systems are used for the basic procedures of isolation and identification of viruses, maintenance of stock cultures and production of vaccines.
Cultivation of Viruses in Laboratory Animals
Animal inoculation helps to confirm the infectious nature of the disease. Laboratory animals used for many kinds of virological works such as clinical studies, pathogenicity of viruses, epizootiology, potency tests on vaccines and production of diagnostic and prophylactic antisera.

An ideal experimental animal is one that is
1. Uniformly and fully susceptible to the virus under study
2. Free from intercurrent infection and adventitious lesions and
3. Inexpensive purchase and maintain
If possible it is better to use germ free, specific pathogen free or colostrums deprived animals for experimental purpose. The same species of animals or atleast closely related species of the natural host is selected for inoculation, wherever possible. For example in case of swine fever, which shows a high degree of host specificity, the same species should be used for inoculation. in others such as in case of rabies with low host specificity, the causative virus will grow in a wide range of animals.

1    Bovines

2    Canines

According to Futter (2001) it is very important the way we cook our food. If the cooking is inadequate it causes food poisoning. While cooking there can be cross contamination starting from raw foods. It includes hands or utensils which can cause food poisoning. Most foods, especially meat, poultry, fish and eggs should be cooked thoroughly to kill most types of food poisoning bacteria. Generally the food should be cooked to a temperature of 75 °C or hotter. When food is cooked, it should be eaten promptly, kept hotter than 60 °C or cooled, covered and stored in the fridge or freezer. Some people are more at risk from food poisoning than others. In order to buy safe food in the market like buying pre-cut produce as a half a watermelon or cut veggies from a salad bar, it is important to choose items that are refrigerated or surrounded by ice. They must be kept in separate bags. FDA (2019) has studied the contamination in lettuce. Shopping at a farmer’s market is a great way to get locally-grown, fresh fruit, vegetables, and other foods. However, safety of these produce needs to be checked. Many markets have their own food safety rules and vendors must comply with them along with the government regulations. Fruits and vegetables must be washed thoroughly under running water just before eating, cutting or cooking. Fruits and vegetables must never be washed with soap or detergent or using commercial produce washes. Even if it has to be peeled before eating, it is still important to wash it first. Any bacteria present on the outside of items like melons can be transferred to the inside when it is cut or peeled. They should be refrigerated within two hours after preparation. The juice or cider must be treated (pasteurized) to kill harmful bacteria. Pregnant women, children, older adults and people with weak immune systems should drink only pasteurized or treated juice. Raw milk can harbor dangerous microorganisms like Salmonella, E. coli, and Listeria. World India (2017) has looked into the food regulations. These can pose serious health risks. Pregnant women, older adults and people with weakened immune systems are at higher risk for illness caused by Listeria. One source for these bacteria is soft cheese made from unpasteurized milk. It is vital to check the label to make sure that it is made from pasteurized or treated milk. Make sure that eggs are properly chilled at the market. FDA requires that untreated shell eggs must be stored and displayed at 45°F. While buying eggs it needs to be ensured that the eggs are clean and the shells are not cracked. Meat should be properly chilled even in the market and kept in closed coolers with adequate amounts of ice to maintain cool temperatures. If it is brought in an insulated bag or cooler during transit it is very beneficial to keep the quality. It should be always kept separately from your other purchases, so that the juices from raw meat (which may contain harmful bacteria) do not come in contact with produce and other foods. Essays (2018) have written a very comprehensive overview of food safety.

1    Ideal surgical pack

2    Nerve blocks

3    Commonly used pre-anaesthetics and anaesthetics

B.anthracis causes anthrax in animals and Wool sorter’s disease, hide porter’s disease, Malignant Pustule in humans. B.cerus causes food poisoning in humans. Other bacillus in this group is non-pathogenic and they are called as anthracoids. B.licheniformis is an emerging pathogen and it is implicated in sporadic abortions in cattle and sheep.
Systematics
Domain : Bacteria
Phylum : Firmicutes
Class : Bacilli
Order : Bacillales
Family : Bacillaceae
Genus : Bacillus
Species : B. anthracis, B. cereus, B. subtilis, B. mycoides,
B. megaterium, B.mesentricus
Family Characters
They are gram +ve large rods, aerobic (facultative anaerobic), endospore forming, capsulated, mostly catalase positive and fermentative organisms. They are motile by peritrichous flagella.
Bacillus Anthracis
History
• Discovery of the anthrax bacillus is credited to Davaine and Rayer (1863 –1868).
• Considerable historic interest attached to anthrax bacilli;

A process control system that identifies the hazardous points in the food production process is known as HACCP (Hazard Analysis Critical Control Point). It puts into place many stringent actions to prevent any hazards from occurring. In any food process if there is strict monitoring and controlling steps, the chances for hazards are minimum. Today, HACCP is an integral part of modern food industry that identifies and controls the major food risks. These are like microbiological, chemical and physical contaminants. These can also be practiced at home scale by checking proper storage, handling, cooking and cleaning procedures. These steps have resulted in industries taking greater responsibility for and controlling the food safety risk factors. This helps in better consumer protection, agriculture, the food processing industries and promotes domestic and international food trade.

The temperature of food storage is important. Below 4⁰C the food pathogens do not grow. Above 60⁰C some colonies begin to die. However, almost all die at 71⁰C. How hot is the food served is important for food safety. The size of the piece is important to how quickly it will cook all the way through. The smaller the piece the faster it will cook. Large or thicker pieces take a longer time to be cooked. Otherwise cooking for a longer time is better so that pathogenic microorganisms do not grow. In case of a thicker piece the color must be checked inside to outside after cooking which should be consistent. If the meat has the bone, check the color/temperature closest to the bone in the thickest part. In case of meat the contamination usually gets introduced while butchering and the practices used. The street food on a cart is perhaps the most contaminated as it has some level of bacteria in it. The meat in the decorated malls also has pathogenic bacteria. The environment plays a very pivotal role which can recontaminate the food even after proper cooking. The amount of harmful pathogens keeps growing in number with the passage of time. Fresh from the grill is the best, an hour or two is not ideal, half a day or overnight could make a person sick. Due to the reason that consumers do not know the time and temperature of cooking it is important to eat food which is freshly prepared.

Mycobacteria are slender rods of varying lengths that sometimes show branching filamentous form resembling ‘fungal mycelium’. Hence, the name mycobacteria, meaning fungus like bacteria.
Although cytochemically gram positiive, the Mycobacteria do not take up the dyes of the gram stain because the cell walls are rich in lipids – Mycolic acid. Once a dye has been taken up by the cells they are not easily decolourised, even by acid-alcohol. Mycobacteria are therefore called as acid-fast bacilli. The genus includes animal and human pathogens as well as saprophytic members often referred to as atypical, anonymous, opportunistic, tuberculoid and MOTT (Mycobacteria other than typical tubercle) bacilli. Clasification of Mycobacteria (Tubercle Bacilli)
I. Slowly growing Mycobacteria
• Mycobacterium tuberculosis causes human tuberculosis in human and dogs.
• Mycobacterium bovis causes bovine tuberculosis in many animal species and also cause tuberculosis in human
• Mycobacterium africanum causes human tuberculosis.
• The human type (Mycobacterium tuberculosis) is primarily a pathogen for man. But can cause disease in cattle, pigs, dogs, monkeys, parrots and other species. The bovine type (Mycobacterium bovis) is a common cause of disease in domestic animal particularly cattle, pigs, cat, dogs and horse. The avian type (Mycobacterium avium) is primarily a pathogen for birds. But can cause disease in cattle, sheep, goat and pigs.

Introduction
Escherichia coli (commonly abbreviated as E. coli) is a Gram-negative, facultative anaerobic, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms (endotherms).
Escherichia coli are common inhabitants of the terminal small intestine and large intestine of mammals. They are often the most abundant facultative anaerobes in this environment. They can occasionally be isolated in association with the intestinal tract of non-mammalian animals and insects. The presence of E.coli in the environment is usually considered to reflect faecal contamination and not the ability to replicate freely outside the intestine.
Morphology
E.coli is Gram-negative, facultative anaerobic and non-sporulating. Cells are typically rod-shaped, and are about 2.0 micrometers (μm) long and 0.25- 1.0 μm in diameter, with a cell volume of 0.6–0.7 μm. It can live on a wide variety of substrates. Strains that possess flagella are motile. The flagella have a peritrichous arrangement. It is motile by peritrichous flagellae, though some strains are non-motile.Spores are not formed. Capsules and fimbriae are found in some strains.
Cultural Characteristics
Escherichia coli or E.coli cells may grow on a solid or in a liquid growth medium under a laboratory condition. Solid and liquid media may have exactly the same composition except that the solid medium contains an extra 1.5% agar. Different E.coli clones may have different properties. Colonies growing on solid media represent different clones. It is an aerobe and a facultative anaerobe. Optimal growth of E. coli occurs at 37°C (98.6°F) but some laboratory strains can multiply at temperatures of up to 49°C (120°F).

Salmonella consists of bacilli leading to Enteric fever, Gastroenteritis, Speticaemia etc. The important member of the genus is Salmonella typhi, which causes Typhoid fever. Salmonella are of two groups;
(i) Enteric fever group consisting of typhoid & Paratyphoid bacilli exclusively or primary human parasites
(ii) Food poisoning group, which are animal parasite but may infect humans causing gastrointestinal infections Salmonella is oxidase negative, catalase positive, indole and Voges Proskauer (VP) negative, methyl red and Simmons citrate positive, H₂S producing andurea negative.
Characteristics
Cells are rod-shaped, non-spore-forming, and predominantly motile by means of peritrichous flagella with diameters of around 0.7-1.5μm and lengths of 2-5μm with a few exceptions. On blood agar, colonies are 2-3mm in diameter. Colonies are generally lactose non-fermenters. They obtain their energy from oxidation and reduction reactions using organic sources, and are facultative anaerobes. They produce acid from glucose usually with the production of gas, and are oxidase negative. Most produce hydrogen sulphide except Salmonella paratyphi A and Salmonella typhi, which is a weak producer. They are identified
with a combination of serological and biochemical tests. Salmonella species are classified and identified into serotypes according to the White-Kauffmann-Le Minor scheme; there are more than 2,500 Salmonella
serotypes that have been described and reported.

Pasteurella and Mannheimia species are small (0.2 x 1-2 μm) non-motile, Gramnegative rods or coccobacilli. They are oxidase-positive facultative anaerobes, and most species are catalase-positive. Although non-enriched media will support their growth, these organisms grow best on media supplemented with blood or serum. They usually remain viable for only a few days on culture plates.
Some species, such as Mannheimia haemolytica, Pasteurella trehalosi and P. aerogenes can tolerate the bile salts in MacConkey agar. In smears from infected tissues stained by the Giemsa method, pasteurellae exhibit bipolar staining.
The family Pasteurellaceae comprises five genera, Actinobacillus, Haemophilus, Mannheimia, Pasteurella and Lonepinella. These genera share a number of common features and some organisms have been reclassified within these genera following deoxyribonucleic acid hybridization studies and 16s rRNA sequencing.
The pasteurellae are small (0. 2 μm by up to 2.0 μm), Gram-negative rods or coccobacilli. They are nonmotile,non-sporing, facultatively anaerobic, fermentative(except for P. anatipesrijer), oxidase-positive andcatalase - positive (except for P. caballi). Althoughunenriched media support their growth, they grow beston media supplemented with serum or blood.
Recent Changes in Nomenclature
Previous name Present name
Pasteurella ureae Actinobacillus ureae
Haemophilus avium Pasteurella avium
Pasteurella pneumotropica Pasteurella dogmatis
(Henriksen biotype)

Introduction

To accomplish experimentations in laboratories, sometimes we need microscope to test/validate our ideas/findings. A microscope is needed to examine objects which are very small which cannot be visualised by naked eyes. However, microscopic study requires some expertise in operating the microscope, particularly the sophisticated microscopes, as well as to prepare (peeling, sections, cutting, fixation, staining, dehydration, mounting, etc.) the sample, so that it can be appropriately examined for the anticipated details.

According to the scientific requirements different types of microscopes are used in the laboratories for research purpose. In this chapter, our aim is to acquaint you with the basics of using and handling of microscopes, particularly a compound microscope that is commonly used by the graduate and post graduate students for basic research.

Microscopy is often designated as the scientific investigation that is employed to review and analyse minute structures and objects using a microscope. The term “microscope” has been derived from ancient Greek words “micros” meaning “small” and “skopeîn” meaning “to look at or see,”.

Microscope

A microscope is a laboratory instrument used to examine objects (biological specimens) which are too small to be seen by the naked eye. The microscope must accomplish three tasks:

• Magnification- to provide a magnified image of the specimen.

• Resolution- to separate the details in the image.

• Contrast- to render the details clearly visible to the eyes.

Although we will tnroduce to you the different types of microscopes used for different speciments, our main focus is to discuss about compound microscope, which is commonly used in the laboratories. Compound microscope was developed around the beginning of 17th century.

Introduction

Test of pollen viability (extent of pollen viability) is important in pollination experiments to ensure fertility/seed setting. It is also important in monitoring state of pollens during storage in ecological/ taxonomic studies as well as during research on pollen biochemistry, genetics, stigma interactions, and incompatibility studies. Pollen viability refers to the ability of pollen to accomplish post-pollination events to achieve fertilization. Pollen viability test has gained importance particularly in plant breeding and hybrid seed production experiments/ practices. In this chapter, you will be acquainted with the basics of pollen viability test. However, for more professional knowledge on the topic readers are suggested to refer specialized books.

Pollen Viability Test

It is important to know the extent of viability of pollens used in pollination experiment, in finding fertility of a plant of interest, to monitor state of pollen on storage, in ecological/taxonomic studies, as well as in research on biochemistry, genetics, stigma interaction, incompatibility assay, etc. of pollen. Several pollen viability tests have been developed time-to-time. It is recommended to initially use different tests for a given pollen type to find out the most appropriate method that provides reliable result on pollen viability. It is important to note that every procedure requires standardization of protocol according to the sample used. Following different tests are used for assessing pollen viability.

1. In-vitro Pollen Germination Test

It is a simple, speedy, and quantitative test for assessing pollen viability. In this method, a collection/sample of pollen is germinated in vitro and observed under microscope. The percentage of germinating pollens (producing pollen tube) after appropriate treatment and a given period of time is determined. The percentage provides an index of viability of the pollen sample.

Globally, antimicrobial resistance (AMR) is an important public health and development threat (WHO, 2020). Being one of the top 10 global public health threats, the ever-increasing rates in the occurrence of drug-resistant bacterial infections have become a grave public health concern for the governments (Laxminarayan et al., 2013). Moreover, such infections would be difficult to treat and therefore can lead to worse clinical outcomes (Laxminarayan et al., 2013). Worldwide, each year about 700,000 people die from antimicrobial resistant infections and the mortality figures are projected to reach 10 million/ year by 2050 (O’Neill, 2016). The crude infectious disease mortality rate in India is more than 400 per 100,000 persons and at least 23,000 deaths in adults and 58,000 neonatal deaths per year are caused by AMR bacteria (Laxminarayan and Chaudhury, 2016). A 2017 estimate puts the cost of developing an antibiotic at around US$1.5 billion (Towse, et al. 2017). Meanwhile, industry analysts estimate that the average revenue generated from an antibiotic’s sale is roughly $46 million per year. “That’s tiny and nowhere near the.