eChapters

Introduction Antimicrobial resistance (AMR) has emerged as one of the gravest threats to modern medicine, undermining decades of progress in infectious disease control. It is cited by the World Health Organization (WHO) as one of the top 10 threats to global health. In 2019 alone, AMR was associated with approximately 4.95 million deaths, including 1.27 million directly attributable to resistant bacterial infections. This makes AMR a leading cause of death worldwide, surpassing HIV/AIDS (0.86 million deaths) and malaria (0.64 million deaths) in the same year. Projections suggest that if the trend continues unchecked, AMR could cause 10 million deaths annually by 2050, representing a profound human-cost escalation. Livestock and Animal burden While the quantitative burden of AMR in animals is not yet globally mapped, its effects in livestock are already both profound and alarming. Over 70% of all antimicrobials globally are used in farm animalsfrequently for growth promotion and disease prevention—not just treatment. This widespread use contributes to emerging resistance in key pathogens—Salmonella, E. coli, Campylobacter in poultry, and severe swine dysentery in pigs. In some outbreaks, swine dysentery, resistant to conventional treatment, has caused up to 90% morbidity and 30% mortality in weaned pigs. Animal-to-Human Transmission and the One Health Paradigm Antimicrobial resistance in livestock doesn’t stay on the farm—it seeps into the environment and crosses over to humans via food chains, direct contact, and environmental pathways. For example, substandard veterinary medicines and antibiotic misuse has been traced in zoonotic infections and veterinary-tohuman AMR transmission.Surveillance gaps remain: the World Organisation for Animal Health (WOAH) acknowledges that while human-related AMR mortality is well-documented, the animal health burden is not yet quantified globally—though numerous projects are underway to address this gap. The failure of conventional antibiotics to treat resistant infections highlights the urgent need for alternative therapeutic strategies. Among the most promising options is bacteriophage therapy—the use of viruses that specifically target and kill bacteria. Bacteriophages Bacteriophages, often referred to simply as phages, are viruses that infect and replicate within the bacteria. They are considered as the most abundant biological entities on Earth, with an estimated amount 10³¹ phage particles present in the biosphere—outnumbering bacteria by approximately tenfold. Phages are naturally found in diverse ecosystems including soil, ocean, sewage, and even within the microbiomes of humans and animals.

Bacteroides spp are non-spore forming gram-negative bacilli that are part of the human resident flora. Microbiologically, they are distinguished from other genera by growth in 20% bile. At present, the Bacteroides fragilis group consists of ten species: B. fragilis (the most frequent isolate), B. distasonis, B. thetaiotaomicron, B. vulgatus, B. ovatus, B. eggerrthii, B. merdae, B. stercoris, B. uniformis, and B. caccae. Since 1990, many organisms previously designated as Bacteroides have been reclassified (see chapter on Anaerobes other than Bacteroides). Bacteroides, the predominant genus in the human
intestine, are important in numerous metabolic activities and may provide some level of protection from invasive pathogens. All 10 species are usually isolated from the colon, although infections caused by or associated with them can include virtually any organ.
Characteristics
The gram-negative Bacteroides spp. or closely related genera are capsulated obligatory anaerobic bacilli that are non-spore forming, pale-staining, and some are motile by peritrichous flagella, while other taxa are non-motile. Bacteroides, Parabacteroides, Odoribacter are generally bile resistant, distinguished from genera which are bile sensitive. They are normally commensal, found in the
intestinal tract of humans (mouth, colon, urogenital tract) and other animals. Many cultures of Bacteroides strains display brown to black pigmentation on blood agar media caused by esculin hydrolysis.
Morphology
In general, all strains were stained poorly with safranin, but well with dilute carbolfuchsin. Motility was not observed in any strains. The morphology of Bacteroides fragilis in fresh cultures and in pus is not
especially characteristic. In this form, the bacillus measures 0.5 by 2.0 to 2.5 micra. Older cultures tend to develop larger forms (0.6 to 0.8 by 2.0 to 9.0 micra) but even in this state the organism cannot be distinguished from aerobic Gram-negative bacilli, by its morphology.

Molecular biology involves the study of DNA, RNA and proteins in cells. It emphasizes the biomolecules that carry out biological processes. One should understand the biomolecules participating in the process to understand the cellular function. In diagnostic microbiology, biomolecules are the analytes for testing and characterization. The analytes for molecular testing are the genomes (DNA), transcriptomes (RNA) and proteomes (Proteins) of the microorganism. Traditional methods of microorganism characterization depend on the culturing of the organisms, which is time-consuming and sometimes challenging to perform. Molecular techniques involve the characterization of DNA, RNA and protein to identify microorganisms. Nucleic acid sequence details are used to classify bacterial species, and mass spectrometry is used to identify microorganisms based on peptide profiles.

DNA (Deoxy ribonucleic acid)
DNA contains the genetic information required for the development and functioning of the living organism. It is the central informational molecule comprising a sequence of A, T, G and C nucelotides linked together by a phosphodiester bond. DNA is double helical in structure, composed of two complementary strands of nucleotides held together by hydrogen bonds between nitrogenous bases.

Molecular diagnostic laboratory set-up has basic requirements of space, laboratory benches and equipments. In terms of laboratory space, minimum four chambers, one each for sample preparation and nucleic acid extraction, PCR reaction mixture preparation, thermal cycling and gel analysis are required. In addition there should be sufficient space for storage of consumables and laboratory wares. In addition, there are some laboratory equipments which are needed in a molecular diagnostic laboratory. In the following section the equipments of a molecular diagnostic laboratory are discussed. The pictures are taken from the instruments present in the laboratory of the author. The purpose of this chapter is to introduce the reader to a molecular diagnostic laboratory. None of the pictures below are meant for promotion of any particular brand or manufacturer.

3.1 UV-VIS spectrophotometer

A spectrophotometer is required for determining the quality and quantity of nucleic acids extracted from a sample. Photometric research applications such as DNA, RNA and protein analysis can be carried out in a UV-Vis Spectrophotometer. There are various units that feature a broad wavelength range (including UV area), path length correction and a fast reading speed. Various instruments also provide an interface that allows quick measurements directly from the instrument or comprehensive easy-to use software for demanding assays.

A. Construction, Use and Care of Light Microscope
Introduction
Microorganism are invisible to naked eyes, hence their detailed study requires a microscope. A microscope can be defined as an optical instrument consisting of a lens or a combination of lenses for getting enlarged or magnified images of minute objects. A simple microscope is essentially an ordinary magnifying lens held on an adjustable stand. Usually it gives magnification up to 20 times the size of the object. A compound microscope consists of two separate lens system – the objective and the eye piece. It gives much greater magnification (100-1500 times) than a simple microscope and is, therefore useful for viewing and examining very minute objects like protozoa, bacteria, yeast, mold and algae which cannot be seen with naked eyes.
The objective of this exercise is to familiarize the students with construction and the use of compound light microscope.
1. Construction of Microscope
A microscope contrite various parts in which principle parts are described below:
i) Frame work: All microscopes have a basic frame structure which includes the arm and base. All other parts are attached to this framework and the base support the microscope.
ii) Stage: It is a horizontal platform that supports the microscopic slide. It has a mechanical stage, which is used for holding and moving the slide around on the stage.

This chapter explains how B-cell receptors, T-cell receptors and major histocompatibility complex molecules participate in antigen recognition. It differentiates MHC class I and class II pathways and describes the processing of endogenous and exogenous antigens. The roles of dendritic cells, macrophages and B lymphocytes as professional antigen-presenting cells are examined. Topics include MHC restriction, antigenic peptides, proteasomal degradation, ubiquitination and recognition by CD4-positive and CD8-positive T lymphocytes. The chapter also discusses superantigens, opsonization, phagocytosis, T-independent antigens and species-specific MHC systems such as BoLA and the avian B complex. These concepts explain the initiation of adaptive immune responses.

Identification of enteric bacilli is of prime importance for controlling intestinal infections. This can be achieved by preventing contamination of food and water by a group of bacteria belonging to family – Enterobacteriaceae which are found in the intestinal tract of human beings and lower mammals. The microorganisms of this family are short, gram negative, non spore forming bacilli. Such common enteric bacteria are a) pathogens such as species of Salmonella and Shigella b) occasional pathogens like species of Proteus and Klebsiella and c) normal intestinal flora like species of Escherichia and Enterobactor.

COMMON FEATURES OF BIOLOGICAL MEMBRANES

Membranes are sheetlike, just a few molecules thick and form closed boundaries between cell compartments.

Membranes contain lipids and proteins, with small amounts of carbohydrayes linked to the lipids and proteins.

Lipids in membranes are small with hydrophobic and hydrophilic portions. Lipid bilayers provide a barrier to the diffusion of polar molecules.

Characteristic functions of membranes are mediated by specific proteins, serving as pumps, channels, receptors, energy transducers and enzymes.

Membrane components associate through noncovalent interactions.

Membranes are asymmetrical, with two sides of the membrane differing from each other.

Lipid and protein molecules often diffuse rapidly in the plane of the membrane.

Central transport of ions and molecules into and out of the cell.

Generate proton gradients for ATP production by oxidative phosphorylation.

Receptors bind extracellular signals and transduce the signal to cell interior.

Introduction

Several hazardous chemicals, microbes, and techniques like recombinant DNA technology are used for research and development work in research Institutes and universities. Therefore, the researchers, technical, helping and supporting staffs as well as the laboratory outputs must be kept free from contaminations. Thus, biosafety is necessary to prevent unintentional exposure/ release of hazardous chemicals, microbes/pathogens, and toxins to the laboratory staff and environment. Following the biosafety guidelines and ethical considerations are essential to maintain safety standards for the researchers as well as the environment. This chapter deals with various biosafety measures and ethical issues to be strictly followed while working in a research laboratory. The required safety level needs to be determined as per the chemical/biological materials are used in the laboratory to keep the researcher/environment safe for sustainable research and developmental activities.

Biosafety

The term biosafety is used to describe the procedure and policies needed to ensure environmental and personal safety. Biosafety refers to the containment principles, technologies and practices that are implemented to prevent unintentional exposure to pathogens, toxins, or their accidental release into the environment. A fundamental objective of any biosafety program is the containment of potentially harmful chemical/biological agents as precautionary safety measures.

Why Biosafety?

With the increasing number of countries adopting molecular biology tools and techniques in life science research and development activities, especially in the areas of agriculture and medicine, the biosafety issues are gaining importance to ensure biological safety for the public and the environment. Conducting research in safe and sustainable manner is not only a personal requirement but essential collective efforts to ensure biological safety for a clean and safe environment. This requires rules, regulations, monitoring bodies, and awareness among the workers as well as the public. Therefore, biosafety per se is an integral part of laboratory research and requires awareness among the researchers so that biological safety can be well taken care from the grassroot level.

to be coccobacillary.They are strict aerobes and do not attack carbohydratesbut derive energy by the oxidation of amino acids. B. avium and B. bronchiseptica are motile by peritricholls flagella but B. pertussis and B. parapertussis are non-motile. All are catalase-positive and oxidase-positive. B. bronchiseptica and B. aium willgrow on MacConkey agar.
The genus Bordetella contains four species, B.pertussis, B.parapertussis, B. bronchiseptica and B. avium. Bordetella pertussis, the type species, and B.parapertussis are human pathogens associated with whooping cough in children. Bordetella bronchiseptica infects a wide range of animal species including man, while B.avium is a pathogen of avian species. The bordetellae are occasional pathogens which have an affinity for ciliated respiratory epithelium.
Bordetella bronchiseptica and B.avium are small (0.2 to 0.5 x 0.5 to 1.5 pm), Gramnegative rods with a coccobacillary appearance. They are catalasepositive, oxidase-positive aerobes and are motile peritrichous bacteria. Because they cannot utilize carbohydrates, they derive their energy mainly from the oxidation of amino acids and have no special growth requirements. They grow on MacConkey agar.
Natural Habitat
The bordetellae are inhabitants primarily of the upper respiratory tract of healthy and diseased humans, animals and birds. B. pertussis and B. parapertussis are human pathogens causing whooping cough and a mild form of whooping cough, respectively. B. bronchiseptica can be present in the upper respiratory tract of pigs, dogs, cats, rabbits, guinea-pigs, rats, horses and possibly other animals. B. avium inhabits the respiratory tract of infected poultry, principally turkeys. Mammalian infections are mainly transmitted by aerosols but in turkeys indirect spread can occur via water and litter.

Borreliae, which are longer and wider than other spirochaetes, have a similar helical shape. In addition to a linear chromosome, which is unique among bacteria, borreliae possess linear and circular plasmids. Although these spirochaetes can cause disease in animals and humans, subclinical infections are also common. Borreliae are transmitted by arthropod vectors.
Usual Habitat
Borreliae are obligate parasites in a variety of vertebrate hosts. Although these organisms persist in the environment for short periods, they depend on vertebrate reservoir hosts and arthropod vectors for long-term survival.
Differentiation of Borrelia Species
Borreliae can be differentiated from other spirochaetes by their morphology, by the low guanine and cytosine content of their genomic DNA and by ecological, cultural and biochemical characteristics. Identification of Borrelia species depends mainly on genetic analysis. At least nine genospecies or genomic groups of B.burgdorferi sensulato, have been identified using DNA-DNA hybridization, 16s rRNA sequencing and other molecular techniques.
Clinical Infections
The species of particular veterinary importance are B. burgdorferi sensu lato, the cause of Lyme disease in animals and humans, and B. anserina which causes avian borreliosis. The significance of two other species, B.theileri andB.coriaceae, as animal pathogens, is uncertain.
Lyme Disease
This condition, also known as Lyme borreliosis, was first identified in 1975 following investigation of a cluster of arthritis cases in children near the town of Old Lyme, Connecticut. The causative agent, a spirochaete, was named Borrelia burgdorferi.

Five genospecies of intestinal spirochaetes have been isolated from pigs namely Brachyspira hyodysenteriae,B. pilosicoli, B. innocens, Serpulina intermedia and S. murdochii. The genera Serpulina and Brachyspira were recently combined. These anaerobic spirochaetes have six to fourteen spirals and are 0.1 to 0.5 μm in width.
Usual Habitat
Pathogenic Brachyspira species are found in the intestinal tract of both clinically affected and normal pigs. Carrier pigs can shed B.hyodysenteriae for up to three months and are the principal source of infection for healthy pigs.
Differentiation of Brachyspira Species
The differentiation of B.hyodysenteriae from other intestinal spirochaetes is based on its pattern of haemolysis on blood agar. Tests for detecting indole production or the hydrolysis of hippurate are also useful diagnostically. Restriction endonnclease analysis, restriction fragment length polymorphism, ribotyping using 16s rRNA analysis, PCR-based assays and multilocus enzyme electrophoresis have been developed both for differentiating species and for distinguishing strains of organisms within species. Brachyspira hyodysenteriae isolates can also he allocated to several serogroups and serotypes.
Pathogenesis
Most information on the pathogenesis of Brachyspira species derives from studies of B.hyodysenteriae. Motility in mucus is an essential virulence factor of thisorganism; mutant strains with altered motility are lesscapable of colonizing the pig intestine. Colonization may be enhanced by factors in mucuswith chemotactic activity for the organisms. Factors with such chemotactic activities have been demonstrated in vitro. Haemolytic activity,demonstrated in vitro, correlates with pathogenicity andthree genes encoding haemolytic and cytotoxic activityhave been cloned and sequenced.

Brucella species are small (0.6 x 0.6 to 1.5 pm), nonmotile, coccobacillary, Gram-negative bacteria. As they are not decolourized by 0.5% acetic acid in the modified Ziehl-Neelsen (MZN) staining technique, they are classed as MZN-positive. In MZN-stained smears of body fluids or tissues, they characteristically appear as clusters of red coccobacilli. For taxonomic purposes, all Brucella species should be classified as Brucella melitensis as DNA hybridization studies have shown that the genus contains only one species.
Brucella species are aerobic, capnophilic and catalase-positive. Apart from B.ovis and B.neotomae, they are oxidase-positive. All Brucella species are urease-positive except B.ovis. Brucella ovis and some biotypes of B.abortus require 5 to 10% CO2 for primary isolation. Moreover, the growth of other Brucella species is enhanced in an atmosphere of CO2. Media enriched with blood or serum is required for culturing B.abortus biotype 2 and B.ovis. Recently, brucellae have been detected in sea-mammals. Smears are made from specimens and stained by the modified Ziehl- Neelsen (MZN) stain. Brucellae appear as small, red-staining coccobacilli in clumps because of their intracellular growth.
Isolation
The brucellae grow well on 5-10 per cent blood agar. However, other than foetal abomasal contents and colostrum, the specimens are likely to contain many contaminating bacteria and fungi, so selective media are usually required. The selective media contain a nutritive blood agar base with 5 per cent sterile seronegative equine or bovine serum and an antibiotic supplement. The antibiotic
supplement used in selective media for B.ovis usually differs from that for B. abortus. Skirrow agar is a satisfactorymedium for both the Campylobacter fetus subspecies and for brucellae, including the most fastidious species such as B. abortus biotype 2, B.canis and B.ovis.

Campylobacter species are slender, curved motile Gram negative rods (0.2 to 0.5 pm wide) with polar flagella. Daughter cells which remain joined have a characteristic gull-winged appearance and long spirals formed by joined cells also occur. These microaerophilic organisms grow best on enriched media in an atmosphere of increased CO2 and decreased oxygen tension. Many Campylobacter species grow on MacConkey agar. They are non-fermentative and oxidase-positive and have variable catalase reactions.
Usual Habitat
Many Campylobacter species are commensals in the intestinal tracts of warm-blooded animals. Campylobacter jejuni and C. lari (formerly C. laridis) colonize the intestines of birds, which can result in faecal contamination of water courses and stored food. A number of Campylobacter species are excreted in the faeces of pigs. Campylobacter fetus subspecies venerealis appears to be adapted principally to bovine preputial mucosa.
Differentiation of Campylobacter Species
Campylobacter species are strictly microaerophilic, requiring an atmosphere of 5 to 10% oxygen and 1 to 10% CO2 for growth. A selective enriched medium such as Skirrow agar is usually used for primary isolation. Differentiation of isolates is based on colonial morphology and certain cultural, biochemical and antibiotic-susceptibility characteristics.
Colonial Morphology
• Campylobacter fetus subspecies venerealis and C.fetus subspecies fetus have small, round, smooth, translucent colonies with a dewdrop appearance.
• Campylobacter jejuni produces small, flat, grey colonies with a spreading, watery appearance.