eChapters

Introduction
The advent of antibiotics, beginning with penicillin in 1941, transformed medicine by rendering bacterial infections treatable [1]. However, antimicrobial resistance (AMR) has emerged as a profound threat, enabling bacteria to survive in the presence of drugs designed to inhibit or kill them and these are rooted in ancient microbial ecosystems evidenced by resistome genes in 30,000-year-old permafrost [2]. AMR has been exacerbated by human practices: approximately 50% of antibiotic prescriptions are inappropriate, 70% of global antibiotics fuel agriculture, and environmental pollution creates resistance hotspots [1]. By 2025, AMR directly causes 1.3 million deaths annually, with economic losses projected at $100 trillion by 2050 [1]. Priority pathogens, including carbapenem-resistant Enterobacteriaceae (CRE), methicillin-resistant Staphylococcus aureus (MRSA), and colistin-resistant strains via mcr-1, challenge last-resort therapies, particularly in low-resource settings [3]. This chapter delivers a technically extraordinary exploration of AMR, structured into genetic, biochemical, and evolutionary mechanisms, each elaborated with molecular precision and step-by-step clarity for learners. Genetic alterations provide the DNA-level foundation, biochemical pathways execute molecular defenses, and evolutionary forces drive adaptation and dissemination. It ensures scientific rigor and broad appeal for students, researchers, clinicians, and policymakers.
Historical Context and Global Burden
AMR emerged shortly after antibiotics’ introduction, with penicillin-resistant S. aureus identified by 1942 due to ß-lactamase production [1]. The 1960s

Introduction 
Antimicrobial resistance (AMR) is one of the most significant global health challenges of the 21st century. Defined as the ability of microorganisms— including bacteria, viruses, fungi, and parasites—to resist the effects of antimicrobial drugs, AMR undermines the progress of modern medicine. Antimicrobials—including antibiotics, antivirals, antifungals, and antiparasitics—are the cornerstone of modern medicine. Their discovery transformed the treatment of infectious diseases and enabled surgical advances, chemotherapy, and organ transplantation. However, Alexander Fleming, the discoverer of penicillin, warned that misuse could render these drugs ineffective. Antibiotics, since their discovery in the 20th century, revolutionized healthcare, saving millions of lives and enabling complex medical procedures. Yet, the misuse and overuse of antimicrobials in human health, veterinary practices, and agriculture have rapidly accelerated the emergence of resistant organisms. Today, this warning has materialized as a global crisis. Antimicrobial resistance (AMR) is now considered one of the top ten public health threats by the World Health Organization (WHO). It is not only a biomedical issue but also a socio-economic and developmental challenge. This chapter aims to provide an in-depth overview of AMR, focusing on definitions, history, mechanisms, causes, global status, impacts, and the urgency of mitigation efforts. It also explores national and global responses and future directions to curb the spread of AMR. AMR is estimated to cause 700,000 deaths annually worldwide, with projections of 10 million deaths annually by 2050 if urgent interventions are not implemented. In addition to health impacts, AMR poses grave threats to food security, sustainable agriculture, economic stability, and global development. This chapter provides a comprehensive overview of AMR, including definitions and conceptual frameworks, historical background, mechanisms of

Dairy animals are the backbone of animal husbandry. India is the largest milk producer and ranks first in the world with a share of 25% of global milk production. Haemoprotozoan diseases pose significant limitations to the health and production of cattle. It leads to significant losses in the livestock industry worldwide. However, most protozoan parasites in the blood are known tocause anaemia by inducing erythro-phagocytosis. In terms of  mortality, reduced milk production and reduced immunity power, hemoprotozoan parasites pose a major threat to the livestock population. Theileria, Babesia, Anaplasma and Trypanosoma are the most important hemoprotozoan diseases of veterinary importance. These diseases affect different livestock species, caused by different species of Trypanosoma, Theileria, Babesia and Anaplasma. Tick-borne hemoprotozoan infections have a significant negative impact on the health and productivity of cattle and result in major losses to the global livestock industry. In tropical and subtropical regions, tick-borne hemoprotozoan infections have a significant impact on animal production. In India, these hemoprotozoan parasites have long posed a serious threat to the survival of exotic and crossbred cattle (Ananda et al., 2009). Haemoprotozoan diseases have deleterious impact on health and production of animals causing death in acute cases, production losses in chronically affected animals which decrease economic share of livestock sector (Bharti et al., 2022)

There are number of internal and external factors that affect the production performance of animals, among which parasites and parasitism are major of concern constraints to animal’s productivity especially in tropical countries. Feed intake and nutrient requirements have been recognized for long time as an important parameter for determining the efficiency of animal production. Most of the parasitic infection in domestic animals is caused by gastrointestinal nematode, which may result in depression in appetite, impairment in GI functions, alteration in protein, energy and mineral metabolism and changes in water balance. Nutrition can affect the ability of the host to cope with the consequences of parasitism and to contain and eventually to overcome parasitism. Since it is proposed that the host gives priority to the reversal of the pathophysiological consequences of parasitism over other body functions, it is to be expected that improved nutrition will always lead to improved resilience. On the other hand, it is proposed that the function of growth, pregnancy and lactation are prioritized over the expression of immunity. Improved nutrition may affect the degree of expression of immunity during these phases and can thus influence the resilience and resistance of host to parasite infection.

Livestock has been essential for Indian agriculture since prehistoric times. Livestock is crucial for tripling farmers’ revenue, for bovines—particularly cattle and buffaloes—play a crucial role in the economy. India has the
largest number of livestock owners, with a wide variety of livestock and poultry breeds that are essential to the socioeconomic advancement of rural communities. The production of consumables and maintaining the country’s food security are inextricably related to the livestock and agricultural sectors. Numerous illnesses have a detrimental effect on the health and productivity of livestock and are highly significant economically. Livestock illnesses have negative effects on producers, costing them money in upkeep, resources, and production.
Protozoal infections are a major contributor to the global burden of infectious diseases and have a profound effect on health, society, and the economy. Nearly one-sixth of the world’s population is at risk of dying from serious diseases like malaria (Plasmodium spp.), African sleeping sickness (Trypanosoma brucei), Chagas’ disease (Trypanosoma cruzi) and various forms of cutaneous and visceral leishmaniasis (Leishmania spp.). Although the burden of protozoal diseases primarily affects tropical and subtropical regions, environmental changes and ecological modifications brought about by both natural and manmade factors have had and are likely to continue to have a significant impact on the emergence and spread of these infections in high-income nations.

Haemoprotozoan parasites are responsible for causing severe infections in humans as well as animals across the world. The important hemoprotozoan diseases of livestock are trypanosomosis, theileriosis, babesiosis, and
anaplasmosis, which are caused by several species of Trypanosoma, Theileria, Babesia, and Anaplasma, respectively, in several species of livestock. These diseases caused huge impact on health and productivity of livestock as well as human beings and causes great economic losses to the livestock production n terms of morbidity and mortality worldwide. In India, the annual economic loss has been esteemed in the livestock due to theileriosis, babesiosis and trypanosomosis of Rs 8426 crore, Rs 4000 crore and Rs 4474 core, respectively. The major clinical signs of the haemoprotozoan disease varies from fever, anorexia, anaemia, abortion, and even death in the acute form of infections. The symptomatological treatment is a quite common practice in the field condition to treat the animals suspected with haemoprotozon infection leading to the development of resistance in animals to currently available drugs against haemoprotozoans. The accurate and prompt diagnosis of haemoprotozoan infection is a key to control these diseases. Routine diagnosis of haemoprotozoan infection has been rely on clinical findings and microscopical demonstration of the infective parasitic stages in the blood or tissue fluid smear. Conventional diagnostic techniques such as Microscopical examination techniques and Immunological assay provide the definite clues about parasitic infections in general, but these tests have some limitations.

The reclassification of the order Rickettsiales was predicated upon comprehensive genetic analyses of 16S rRNA, groESL, and surface protein genes. Consequently, organisms within this order were systematically assigned to one of two families: Anaplasmataceae and Rickettsiaceae. Within the Anaplasmataceae family, phylogenetic analyses consistently delineated four genetically distinct clades: (1) Anaplasma, (2) Ehrlichia, (3) Wolbachia, and (4) Neorickettsia. Despite the obligate intracellular nature of rickettsial organisms in both families, those classified under Rickettsiaceae proliferate freely within
the host cell cytoplasm, whereas members of the Anaplasmataceae family are confined exclusively to membrane-bound vacuoles within the cytoplasm of host cells.
The first documented cases of anaplasmosis trace back to the late 19th century in South Africa. However, it wasn’t until 1910 that the causative agent of the disease was conclusively identified morphologically as “marginal points” within red blood cells by Max Theiler, later named it Anaplasma marginale. A year later, he identified another species, A. centrale, which caused a milder form of the disease in cattle. During the 1930s, Sanborn, Stiles, and Moc demonstrated the mechanical transmission of A. marginale by horse flies and stable flies. In the late 1980s, the life cycle of A. marginale was fully elucidated in ticks belonging to the genus Dermacentor.

Bovine babesiosis is a significant febrile disease, primarily affecting cattle and buffalo, transmitted by ticks. The disease is marked by the rapid proliferation of parasites within the bloodstream, leading to the extensive destruction of red blood cells. This results in a cascade of clinical symptoms, including anemia, jaundice, hemoglobinuria, and splenomegaly, with the potential for severe cases to culminate in death. The acute form of bovine babesiosis is particularly severe, often characterized by these pronounced symptoms, which can lead to rapid deterioration in the affected animals. Babesiosis is not merely an acute illness but can also persist as a chronic infection. Chronic bovine babesiosis is typified by ongoing anemia and varying degrees of weight loss, reflecting the enduring impact of the parasite within the host. Economically, this disease poses a formidable challenge, especially in regions where cattle are most susceptible. Since the first description of Babesia bovis by Victor Babes in Romania in 1888, numerous distinct species have been identified, each contributing to the global burden of this disease (Hunfeld et al., 2008). The impact of bovine babesiosis on livestock improvement efforts in endemic regions is profound. The introduction of susceptible, naive animals into these areas often leads to significant losses, as these animals are highly vulnerable to the disease.

Theileriosis is a tick-borne protozoan disease that affects a wide range of domestic and wild animals, particularly cattle. While multiple species of Theileria exist, Theileria parva and Theileria annulata are the most important pathogens in terms of their impact on domestic cattle. This disease is transmitted by ixodid ticks and has a significant impact on cattle populations in tropical and subtropical regions. Theileriosis poses a substantial threat to livestock productivity, affecting economic stability in many countries, especially those that rely heavily on agriculture and cattle farming.
The disease has two primary forms: East Coast fever (ECF), caused by T. parva, which predominantly affects cattle in eastern, central, and southern Africa; and tropical theileriosis, caused by T. annulata, which affects cattle across northern Africa, the Middle East, and parts of Asia. Both diseases are highly pathogenic and, if left untreated, can lead to high morbidity and mortality rates, significantly impacting livestock production.

Kingdom: Protozoa
Phylum: Euglenozoa
Class: Kinetoplasta
Order: Trypanosomatida
Family: Trypanosomatidae
Genus: Trypanosoma
Members of the genus Trypanosoma are responsible for trypanosomosis in animals and human beings and are found in the blood vascular system and tissues all over the world. All the species of trypanosomes are transmitted biologically or mechanically through an arthropod vector except T. equiperdum, which is transmitted through coitus in equines.

Protozoa are considered as unicellular eukaryotic organisms and evolved after bacteria and thousands years early to multi-cellular organisms as well as animals. This unicellular eukaryotic organism contains all the organelles or structures like multi-cellular organisms but in single cells. Similar to multicellular, the genetic information of protozoa is also stored in chromosomes. The protozoa differ from bacteria in different manners. The differentiating features of bacteria and protozoa are given in Table 1.
1. General Structures of Protozoa
Protozoa, like other eukaryotic cells, contain a nucleus, endoplasmic reticulum, mitochondria, Golgi apparatus, and lysosomes. Beyond these common organelles, protozoa feature a range of unique structures with specific functions that enable them to operate independently. Protozoan cells can be categorized into two primary components: the cytoplasm and the nucleus.

This chapter outlines fundamental molecular biology protocols essential for genomic analysis and molecular characterisation. Modern molecular biology relies heavily on precise analytical techniques for studying genetic material. Hence, this section presents a typical workflow encompassing nucleic acid extraction, amplification, and subsequent analysis. These methodologies are fundamental to various applications in molecular biology, including phylogenetic analysis, gene expression studies, and molecular diagnostics.
The chapter covers four core molecular biology and bioinformatics techniques, each building upon the previous:
1. Genomic DNA Extraction: Protocols for efficient isolation of DNA from biological samples. This encompasses cell lysis, protein denaturation, and nucleic acid purification through standard separation techniques. The quality and purity of extracted DNA significantly influences downstream applications.
2. Primer Design with Primer3: In silico design of oligonucleotide primers for PCR amplification. This section covers primer design parameters including optimal melting temperature (Tm), GC content, primer length, and considerations for avoiding secondary structures and primer-dimer formation.

This chapter explores the essential techniques used in veterinary parasitology to collect, identify, and diagnose parasitic infections. These methods are crucial for understanding general animal health, the complex relationships between parasites and their hosts and for tracking disease outbreaks, that will ultimately allow the development of effective control strategies. Whether in a research lab or a clinical setting, the techniques discussed here are widely used to detect and study parasites that impact both domestic and wild animals.
2.1 Collection of Specimens for Parasitological Examinations
Different species of parasites affects almost all parts of the host. These organisms can inhabit virtually every organ system of the host animal, from the brain and lungs to the digestive tract and skin. Therefore, a thorough examination of both living animals and post-mortem cases is essential for collecting parasites and determining cause of death (when applicable). When collecting from live animals, samples should be collected after through exanimation of the animals and following the standard procedure. The collection of correct samples, use of appropriate transport media and proper handling of specimens are crucial factors that directly influence diagnostic accuracy.
The type of specimen collected depends on the methods of identification and diagnosis. For example, diagnosis of blood parasites requires specific types of samples: peripheral blood smears for microscopic examination of hemoprotozoans, serum or plasma for ELISA and other serological tests, and unclotted whole blood for molecular diagnostic methods such as PCR and qPCR.

This chapter introduces the essential components of laboratory work that form the foundation for veterinary parasitology and molecular biology research. A well-equipped laboratory along with proper safety protocols and correctly prepared reagents, is the key to reliable experimental outcomes and a secure working environment.
The following sections describe the standard operating procedures and laboratory biosafety, standard equipment used in research work, and the fundamental principles of solution preparation. These basic laboratory skills are essential prerequisites for the specialised techniques covered in subsequent chapters on veterinary parasitology, molecular biology and bioinformatics. Understanding and applying these foundational concepts allows researchers to maintain experimental consistency, ensure reproducible results, and conduct investigations safely and efficiently. The methods and principles outlined here will be regularly applied throughout the various techniques detailed in this manual, serving as essential knowledge for all laboratory personnel as well short-term visitors.
1.1 Laboratory Standard Operating Procedures and Safety
Laboratory safety is fundamental to protecting personnel, maintaining sample integrity, and ensuring reliable experimental outcomes. This section outlines essential laboratory conduct, safety protocols for personnel and equipment, waste management, and emergency protocols for standard laboratories carrying out diagnostics as well as research.

Emerging Infectious Diseases
Emerging infectious diseases are those due to newly identified and previously unknown infections which cause public health problems either locally or internationally.
Re-emerging Infectious Diseases
Re-emerging infectious diseases are those due to the reappearance and increase of infections which are known, but had formerly fallen to levels so low that they were no longer considered a public health problem.
What Causes Emergence or Re-emergence of Infectious Diseases?
Several factors contribute to the emergence and re-emergence of infectious diseases, but most can be linked with the increasing number of people living and moving on earth: rapid and intense international travel; overcrowding in cities with poor sanitation; changes in handling and processing of large quantities of food; and increased exposure of humans to disease vectors and reservoirs in nature. Other factors include a deteriorating public health infrastructure which is unable to cope with population demands, and the emergence of resistance to antibiotics linked to their increased misuse.

Trans-boundary Animal Diseases (TADs) are highly contagious diseases of livestock in the world and transmissible diseases which have the potential for very serious and rapid spread, irrespective of national borders, which has serious socio-economic or public health consequence and they are importance in the international trade of animals and animal products. With rapidly increasing globalization, an associated risk of movement of trans-boundary diseases is emerging. Trans-boundary animal diseases represent a serious threat. They reduce production and productivity, disrupt local and national economies and also threaten human health (zoonotic). Trans-boundary animal disease is a concern globally; cumulative effort is needed at international level to minimize the spread of infectious diseases across the borders.
Considering that livestock rearing constitutes a significant share in the national economy of a developing country, it is imperative to take up disease control initiatives. Measures are required to safeguard the livestock industry from epidemics of infectious diseases and to uphold safe international trade of  livestock and their products. In this regard, it is essential to develop scientific and risk-based standards that facilitate the international trade in animal commodities.
Introduction
Trans- boundary Animal Diseases (TADs) are highly contagious diseases of livestock in the world. Moreover, their economic importance is a major constraint in international trade. Their implication on human Health and National food security cannot be over emphasized. Zoonotic diseases among TAD’s include diseases like West Nile Virus (WNV), Rift Valley Fever (RVF), Mad Cow disease (BSE), Bovine Tuberculosis and Highly Pathogenic Avian Influenza (HPAI). Other important TADs are Foot and Mouth Disease (FMD), Contagious Bovine Pleuropneumonia (CBPP), Lumpy Skin Disease, African
Swine Fever (ASF) and Newcastle Disease (ND). They have the potential for very rapid spread, irrespective of national borders and these diseases can cause serious socio-economic and possibly public health consequences.

Emerging Infectious Diseases
Emerging infectious diseases are those due to newly identified and previously unknown infections which cause public health problems either locally or internationally.
Re-emerging Infectious Diseases
Re-emerging infectious diseases are those due to the reappearance and increase of infections which are known, but had formerly fallen to levels so low that they were no longer considered a public health problem.
What Causes Emergence or Re-emergence of Infectious Diseases?
Several factors contribute to the emergence and re-emergence of infectious diseases, but most can be linked with the increasing number of people living and moving on earth: rapid and intense international travel; overcrowding in cities with poor sanitation; changes in handling and processing of large quantities of food; and increased exposure of humans to disease vectors and reservoirs in nature. Other factors include a deteriorating public health infrastructure which is unable to cope with population demands, and the emergence of resistance to antibiotics linked to their increased misuse.
Avian Influenza
Avian influenza is a zoonotic, globally important disease of birds that can be categorised as either low pathogenic (LPAI) or highly pathogenic (HPAI) according to the virulence of the virus in animals. Outbreaks of LPAI are common around the world but LPAI typically causes no clinical signs or only minor illness in infected birds. LPAI strains are generally less of a threat to human health; in addition, LPAI can have significant economic repercussions as a result of export restrictions and culling of birds, particularly in developing countries where the use of vaccines and other veterinary medicines is difficult
due to weak veterinarian services and small farming units. Compared with LPAI, HPAI is more readily detected in poultry due to very high levels of mortality in infected birds, and can cause potentially catastrophic economic consequences, from significantly reduced livestock populations to lost export markets. Though relatively rare, sporadic human infections of HPAI have occurred in cases of close contact with infected birds and caused serious illness
and even death.

Chlamydiae are obligate intracellular bacteria with an unusual developmental cycle during which unique infectious forms are produced. They replicate within cytoplasmic vacuoles in host cells. On account of their apparent inability to generate ATP, with resultant dependence on host cell metabolism, they have been termed ‘energy parasites’.
The family Chlamydiaceae belongs to the order Chlamydiales. Currently two genera, Chlamydia and Chlamydophila, and nine species are described. Formerly a single genus and four species, Chlamydia trachomatis, C. psittaci, C. pneumoniae and C. pecorum, were recognised. This classification was based on phenotypic characteristics such as host preference, inclusion morphology, iodine staining for the presence of glycogen, and sulphonamide susceptibility. However, recent nucleic acid sequencing studies of the 16s and 23s rRNA genes confirm two distinct lineages.
In the developmental cycle of chlamydiae, infectious and reproductive forms are morphologically distinct. Infectious extracellular forms, called elementary bodies (EBs) are small (200 to 300 nm), metabolically inert and osmotically stable. Each EB is surrounded by a conventional bacterial cytoplasmic membrane, a periplasmic space and an outer envelope containing lipopolysaccharide.
The periplasmic space does not contain a detectable peptidoglycan layer and the EB relies on disulphide cross-linked envelope proteins for osmotic stability. Elementary bodies enter host cells by receptor-mediated endocytosis. Acidification of the endosome and fusion with lysosomes are prevented by mechanisms which are not fully understood. A process of structural reorganization within the pathogen, of several hours duration, results in the conversion of an EB into a reticulate body (RB). The RB, about 1 μm in diameter, is metabolically active, osmotically fragile and replicates by binary fission within the endosome. The endosome and its contents, when stained, is called an inclusion. When a number of inclusions containing RBs of C.trachomatis are formed in an infected cell, fusion of these structures may
occur.

Organisms in the order Rickettsiales form a diverse group of small (0.3 to 0.5 x 0.8 to 2.0 pm), non-motile, pleomorphic Gram-negative bacteria which replicate only in host cells. They can be cultured in the yolk sac of embryonated eggs or in selected tissue culture cell lines. Because they stain poorly with aniline dyes, these organisms should be stained by Romanowsky methods such as Giemsa or Leishman. In addition to host-cell dependence and poor affinity for basic dyes, a requirement for an invertebrate vector distinguishes them from conventional bacteria and the Chlamydiales.
Epidemiology
Animal hosts and arthropod vectors are the reservoirs for most rickettsiae. A number of rickettsia1 organisms, including Ehrlichia canis, Anaplasma marginale and Haemobartonella felis produce latent infections. In arthropods, rickettsiae replicate in the epithelial cells of the gut before spreading to other organs, including the salivary glands and ovaries where further replication may occur.
Organisms are transmitted when the arthropod feeds on the animal host. Some organisms such as Rickettsia rickettsii are maintained in a tick population by transovarial transmission. Trans-stadial but not transovarial transmission of E. canis and E. phagocytophila occurs in ticks. With the exception of Coxiella burnetii, which produces endospore-like forms and can remain viable in dust for up to 50 days, most rickettsiae are labile outside host cells. Aerosol transmission of C.burnetii commonly occurs in domestic animals and humans. In addition, a silent cycle involving ticks and small wild mammals may constitute a possible source of infection for some domestic species.
Pathogenesis and Pathogenicity
Many Rickettsia species including the causal agents of typhus (R. prowuzekii), murine typhus (R. typhi) and scrub typhus (R. tsutsugamushi) are primarily human pathogens. Rocky Mountain spotted fever, caused by Rickettsia rickettsii, which is a common rickettsia1 disease of humans, also affects dogs.

Bacteria from the genus Anaplasma belong to the Procaryota kingdom, the family Anaplasmataceae. Anaplasma belong to obligate intracellular microorganisms, Gram-negative bacteria, living in the blood cells of mammals. Vertebrates can be their reservoir, i.e. an environment where the pathogen can live and proliferate for many years. However, in many cases bacteria from the genus Anaplasma cause diseases in domestic animals and people.
Taxonomical Position of Bacteria from the Genus Anaplasma
The genera Anaplasma, Ehrlichia, Neorickettsia and Wolbachia include obligate intracellular bacteria, parasitizing in the vacuoles of cells in eukarytic hosts. Animal pathogens were attributed to the genus Anaplasma, such as A. centrale, A. marginale, A. platys, A. ovis and A. bovis, and also the aetiological factor of human anaplasmosis, A. phagocytophilum.
Anaplasma marginale
A. marginale are obligate intracellular bacteria parasitizing in erythrocytes of higher vertebrates, mostly ruminants.
Anaplasma Centrale
A. centrale is a parasite in the erythrocytes of ruminants, mainly cattle. As opposed to A. marginale, it creates concentrations in the central part of the cell. A. centrale is less pathogenic to cattle than A. marginale but, most importantly, occasionally gives resistance against the latter. Hence it is used for the preparation of live vaccine strains, assuring immunological protection against bovine anaplasmosis.
Anaplasma Bovis
A. bovis is a bacterium detected mainly in cattle, but also observed in small mammals which are probably a reservoir of this bacterium. It occurs in monocytes, and the disease it causes is called monocytic anaplasmosis. The symptoms of the disease are most visible in calves, but also in adult animals; symptoms include weakening of the body, marked reduction in weight, elevated temperature, enlargement of prescapular lymph nodes, paling of the mucous membranes, and in many cases an elevated amount of secreted mucus.