eChapters

Metabolites are small molecules generated or utilized during cellular metabolism and include amino acids, carbohydrates, lipids, organic acids, nucleotides, vitamins, hormones, and secondary metabolites. This chapter introduces the major classes of metabolites and their biological significance. It explains metabolic architecture as the organization and interaction of interconnected biochemical pathways that regulate cellular energy production, biosynthesis, degradation, signaling, and adaptation. The chapter describes primary and secondary metabolites and examines central metabolic pathways and their interrelationships. Understanding metabolic architecture provides the foundation for interpreting metabolomic profiles and identifying changes associated with physiological conditions, environmental stresses, disease processes, and alterations in cellular metabolic regulation.

Before proceeding to find out the nature of the egg content of the faecal sample, one should know what class of animal the faecal sample belongs to. It is easy to identify a faecal sample of herbivore from that of an omnivore or a carnivore. The dung of cattle, sheep and horse will contain lot of vegetable fibers. The colour of the dung under normal conditions will be dark greenish yellow with a peculiar aromatic smell. But the faeces of a dog or cat will be soft and pasty devoid of fibers and dark yellow in colour with an unpleasant smell. Several procedures commonly used to examine faeces for parasites are described in this section. Before attempting to apply these procedures, a few points should be considered:

Lignieres and Spitz first isolated Actinobacillus from the cattle with actinomycosis like condition in Argentina in 1902. The findings were also confirmed by Nocard in 1902. Later the isolated bacterium from the cattle was named as Actinobacillus lignieresii in memory of Lignieres.

Pfeiffer (1892) first isolated Haemophilus influenzae as a cause of human influenza and it is also known as ‘Pfeiffer’s bacillus’. Other workers isolated and described different species of the genus Haemophilus from animals and birds. In 1903, Friedberger, a research assistant of Pfeiffer, isolated similar kind of bacteria from prepuce of dogs which was described later as Haemophilus canis. In 1925, Mitchell isolated Haemophilus ovis from the respiratory tract of sheep. Lewis and Shope (1931) isolated and illustrated Haemophilus suis from the infected pigs. Glasser found the association between fibrinous serositis, polyarthritis in pigs (Glasser’s disease) and a small gram-negative rod as early as 1910, later became known as Haemophilus parasuis. The bacteria was first isolated by Hjarre and Wramby in 1943. De Blieck (1931) isolated a hemophilic bacterium from the birds suffering with ‘infectious coryza’ and named the bacteria as Bacillus haemoglobinophilus coryzae gallinarum. Later it became known as Haemophilus paragallinarum (Avibacterium paragallinarum).

Cohn (1875) first observed and described a branching filamentous microorganism in the ‘concretions’ (coagulated mass) taken from a lacrimal canal, later identified as Actinomyces israelii. Bollinger first reported the yellow granules in jaw tissues of the cattle in 1877 and established the significance of the bacteria as the etiological agent. In 1878, Israel described the first human case. In 1879, Hartz observed the microscopic appearance of granules of Actinomyces and suggested the name Actinomyces bovis. Mosselman & Lihnaux (1890) and Wolf & Israel (1891) first isolated the bacteria in artificial laboratory media from lumpy jaw cases of cattle and human actinomycosis, respectively.

In 1850, Bacillus anthracis was first reported by three individuals in two different countries i.e. Delafond and Davaine in France, and Pollender in Germany. They could not establish the relationship of the bacterium with the infection. Absolute proof of the anthrax etiology as B. anthracis was established by Robert Koch during 1876-77. The name Bacillus anthracis was first coined by Cohn (1875) and the disease ‘anthrax’ was first named by Hippocrates (5th century B.C.). In Greek language meaning of ‘anthrax’ is ‘coal’ referring to the black coloured skin or blood in affected animals.

Guillame de Baillou (1673), a physician, described an outbreak of recurrent severe cough that could induce vomition and suffocation in human in Paris. The infection was described as ‘Quintan^tussis which was later confirmed as ‘whooping cough’. The causative agent Bordetella pertussis was discovered by Jules Bordet and Octave Gengou in 1906 in Paris. The Gram-negative bacilli were isolated from sputum of Jules Bordet’s son. Bordetella bronchiseptica was first isolated by Ferry (1911) and M’Gowan (1911) who considered the bacteria as the cause of distemper in dogs. Later B. bronchiseptica was known as primary cause of ‘kennel cough’ infection along with the Canine Adenovirus and Canine Parainfluenza virus. Moreno- Lopez named the bacteria B. bronchiseptica.

The genus Moraxella was named after Morax who first isolated the bacteria from human conjunctivitis in 1896.

Clinical conditions similar to brucellosis have been described since the time of Hippocrates (450 BC). J. A. Marston (1861) first described brucellosis as gastric remittent fever in Malta, the Southern European island country. Sir David Bruce (1886) first isolated small coccobacilli from an infected soldier’s spleen and purified the culture in 1887, and named Micrococcus melitensis. Later the genus was renamed as Brucella in his honour. Similarly, Bang and Stribolg (1897) first isolated Brucella abortus from abortion material of a cow. Traum in 1914 isolated Bacillus abortus suis, currently known as Brucella suis. In 1953, Brucella ovis was firs isolated in New Zealand by Budlle and Boyes from the aborted material of sheep. Carmichael and Bruner (1968) first isolated B. canis from the aborted placenta of a dog.

In India first report of ‘contagious abortion’ associated with Brucella was found in annual report (1918) of imperial veterinary research institute (presently Indian veterinary research institute), Mukteswar, Uttarakhand and later in 1942 by Polding.

The word ‘chlamydia’ comes from the Greek word ‘chlamys’ means ‘cloak draped around the shoulder’. It describes how the intracytoplasmic inclusions produced by the bacterium appear under the microscope(‘draped’ around the host cell nucleus). Chlamydia was first discovered by a Czech zoologist and parasitologist, Stanislaus von Prowazek in 1907 in Berlin. Originally because of its structure it was thought to be a virus. In 1963 it was recognized as a bacterium rather than a virus.

Properties of genus

1. Morphology: Chlamydia are Gram-negative, non-motile, pleomorphic and obligate intracellular bacteria. The Gram-negative cell wall consists of cytoplasmic and outer membrane. However, they lack peptidoglycan layer. Moreover, they also encode major outer membrane protein (MOMP), the key antigenic determinant used for serological differentiation of chlamydiae.

They are poorly stained by Gram’s stain. Chlamydiae are readily stained by Castaneda (blue in appearance), Giemnez and Macchiavello (red in appearance) methods. However, Giemsa staining is preferable to stain the inclusions (chlamydial micro colony) in the cell culture. Inclusion bodies of the Chlamydiae are basophilic in nature. The staining of the inclusion bodies may differ depending on the species. In C. trachomatis, the inclusion bodies contain glycogen matrix and can be easily stained with iodine solution.

The genus Clostridium belongs to the family Clostridiaceae. The genus name was originated from the Greek word ‘klo ster’, which means ‘spindle’ referring to their individual rod shape. Clostridia are gram positive, large, sporulating anaerobic rods (except C. piliforme which is gram negative). At present, there are more than 200 numbers of species under the genus Clostridium. Among them, 15 species can produce potent toxins and are considered as pathogenic for animals and human. The animal pathogenic clostridia can be classified as neurotoxic, histotoxic and enteric.

Corynebacterium diptheriae was first observed by Klebs in 1883 and was described in details by Loeffler in 1884. So it is also known as ‘Klebs- Loeffler bacillus’. In 1888, Nocard isolated a similar type of bacteria from cases of bovine farcy. In 1894, Preisz described it completely and revealed its similarities with the diphtheria bacilli. Later it was named as Corynebacterium pseudotuberculosis (Preisz-Nocard bacillus). Bollinger (1890) first reported C. renale from the cases of pyelonephritis in cows.

The genus Trueperella was proposed by Yassin and others (2011) to describe T. pyogenes previously known as Arcanobacterium pyogenes or Actinomyces pyogenes or Corynebacterium pyogenes. The genus Arcanobacterium was first described (Collins and others, 1982) to include the bacteria originally isolated as Corynebacterium haemolyticum from the wounds of American soldiers. On the basis of 16S rRNA comparisons and menaquinone and phospholipid compositions it is proposed that the species Arcanobacterium pyogenes should be reclassified as members of the new genus Trueperella.

The genus Rhodococcus was first introduced by Zopf (1891) as a soil bacterium used in horticulture. Magnusson (1923) described C. equi (Rhodococcus equi) from suppurative pneumonia of a foal.

The family Enterobacteriaceae belongs to the order Enterobacterial. The family comprises 25 genera such as Escherichia, Salmonella, Klebsiella, Yersinia, Proteus, Providencia, Shigella, Enterobacter, Morganella, Edwardsiella, Budvicia, Cedecea, Citrobacter, Ewingella, Hafnia, Kluyvera, Leclercia, Leminorella, Moellerella, Pantoea, Rahnella, Tatumella, Trabulsiella, Serratia and Yokenella, out of which the first 10 genera are consistent pathogenic in humans, animals and birds.

Escherichia coli

Escherichia coli were first isolated by Theodor Escherich in 1885 from the faeces of infants. It was named in honour of the German pediatrician and its major natural habitat (colon). Previously it was also known as Bacillus coli or Bacterium coli (B. coli).

Properties

1. Bacteroides, Dichelobacter and Fusobacterium are major gram negative anaerobes isolated from human and animals. Other gram negative anaerobes are listed below (Table-1).

2. The bacteria are tolerant to oxygen in comparison to strict anaerobes. The bacteria do not die after oxygen exposure.

3. The bacteria naturally inhabit the intestine and mucosal surfaces of human and animals. Majority of them are opportunistic pathogens. Often they cause mixed bacterial infection along with aerobes. The aerobes remove oxygen and create an anaerobic condition favourable for their growth. The aerobes may also supply other growth requirements. For e.g. Porphyromonas asaccharolytica requires vitamin K for its growth, supplied by another aerobic bacterium, not the host.

Antony Van Leeuwenhoek (1681) first observed the spirochetes under the microscope and described them. In 1833, Ehrenberg used the name Spirochaeta for free living spiral shaped organisms. Obermeir (1873) first described a spirochete of pathogenic significance isolated from human patients with relapsing fever. Inada (1916) first isolated Leptospira icterohaemorrhagiae from human patients of Weil’s disease. In 1980, Nogochi proposed the name as Leptospira.

In India, Taylor and Goyale (1931) first reported Leptospira from human patients suffering with jaundice in Andaman Islands. Ayyar (1932) first reported Leptospira from dogs in Chennai city.

Murray (1926) and Pirie (1927) first described Listeria independently. In India, animal listeriosis in sheep was first reported by Mahajan (1936) in Andhra Pradesh.

Robert Koch first isolated Erysipelothrix in 1876 from a mouse he inoculated with putrefying blood. Koch initially named the bacterium “Bacillus of mouse septicemia”. Pasteur and Dumas (1882) first isolated Erysipelothrix from the infected pigs. Friedrich Loffler (1886) first experimentally produced the infection in pigs. Rosenbach (1909) first described human erysipeloid. The nomenclature of the bacterium is changed several times and Erysipelothrix rhusiopathiae was finally accepted by the scientific community in 1966. The name is originated from Greek words such as ‘erysipelas’ (red skin), ‘trix’ (hair), ‘rhusius’ (reddening) and ‘pathus’ (disease).

In 1882, Robert Koch first isolated the bacterium, stained with alkaline methylene blue and vesuvin as counter stain and showed its etiologic relationship with tuberculosis. Ehrlich first developed acid-fast stain in 1882 and used for better staining of the bacteria which was adopted by Robert Koch. The acid-fast staining technique was modified later by Franz Ziehl and Friedrich Neelsen, accordingly, it is known as Ziehl-Neelsen (Z-N) staining. In 1890, Koch prepared a glycerine broth extract of the tubercle bacillus, known as ‘tuberculin’. He tried to prepare it as a curative agent; but it was used later in diagnosis. In 1895, Johne and Frothingham described the first case of paratuberculosis in cattle which became known as ‘Johne’s disease’. The causative bacterium was later isolated by Twort and Ingram in 1910.

In India, bovine tuberculosis is reported from several states since 1959 (Dhanda and Lall, 1959). The bovine paratuberculosis in India was first reported by Nanda and Singh (1940) from Haryana.

The word ‘Mycoplasma’ is originated from the Greek word mykes (fungus) and plasma (formed). It was first used by Albert Bernhard Frank (1889), a German biologist. He thought it was a fungus. An older name for Mycoplasma was pleuropneumonia-like organisms (PPLO), referring to organisms similar to the causative agent of contagious bovine pleuropneumonia (CBPP).It was later found that the fungus-like growth pattern of Mycoplasma mycoides is unique to the species. It was first isolated in 1898 by Edmond Nocard and E. Roux from bovine lungs. Mycoplasma pneumoniae was considered as a virus previously. Eaton and his co-workers cultured the causative agent of human primary atypical pneumonia (PAP) or ‘walking pneumonia’. It was known as ‘Eaton’s agent’. In the early 1960s, Leonard Hayflick, in Philadelphia, was first to isolate and cultivate the causative agent of primary atypical pneumonia and identified the agent as a Mycoplasma pneumoniae.

Properties of genus

1 Morphology: Mycoplasma have no cell wall and therefore are resistant to the antibiotics used to inhibit the cell wall synthesis like penicillin, cephalosporin and vancomycin. The bacteria are the smallest prokaryote (0.3-0.8µm). They are pleomorphic due to absence of the rigid cell wall. The different shapes include ring, globules, filaments and elementary bodies. Due to their plasticity in shape and size, they can be passed through the membrane filter with pore size of 0.45µm. The cell membrane is constituted of trilaminar structure enriched with phosphoprotein, lipoprotein, glycolipid, phospholipid and sterol. So Mycoplasma require sterol for their growth.

Edmond Nocard (French veterinarian) first isolated filamentous aerobic bacteria from clinical cases of ‘bovine farcy’ in 1888. In 1889, the genus Nocardia was formed by Trevisan and the bacterium originally described by Nocard was named as Nocardia farcinica. In 1891, another similar bacterium (Nocardia asteroides) was isolated by Eppinger from a human brain abscess. Initially it was thought that they are the same bacteria. On the basis of the genetic differences the separate entity was established later.

The re-examination of Nocard’s original isolate revealed the existence of two different filamentous bacteria i.e. Mycobacterium and Nocardia. The cause of ‘bovine farcy’ has been subsequently attributed to Mycobacterium. Later, it is proved that Mycobacterium farcinogenes is the actual causative agent of ‘bovine farcy’.

Louis Pasteur (1881) first isolated the bacterium from fowl cholera outbreak and attenuated the bacteria to develop a vaccine for the chicken. Trevisan (1887) coined the generic name ‘Pasteurella’ in honour of Louis Pasteur. Lignieres (1900) proposed the name of the species according to the animals acted as the major hosts such as Pasteurella aviseptica for fowls, P. suiseptica for pigs, P. boviseptica for bovines, P. oviseptica for ovines and P. leptiseptica for rabbits. Rosenbuch and Merchant (1939) proposed the name of single species Pasteurella multocida which is in use till date.

Mannheimia is an exclusive animal pathogen and is associated with ‘shipping fever’ or bovine respiratory disease (BRD) in cattle. Kitt (1885) first described the bacterium as Bacterium bipolare multocidum isolated from shipping fever in cattle. The nomenclature was further changed into Bacillus boviseptica and finally into Pasteurella haemolytica. Based on fermentation capability of arabinose and trehalose, P. haemolytica was subdivided into two biotypes (A and T) and several capsular serotypes. The biotype A (with 12 capsular serotypes) was classified under a new genus Mannheimia due to disparities in 16S rDNA sequence, cultural and pathological characteristics.

Lucke (1862) first described that the bluish green discolouration of surgical dressing is associated with the presence of small vibrio like organisms. Gessard (1882) first isolated the bacteria as Pseudomonas.

Identification of the etiologic agent of glanders was performed in 1882 by isolating a bacterium from the infected liver and spleen of horse. Since its discovery, the pathogen has been classified as Loefflerella mallei, Pfeifferella mallei, Malleomyces mallei, Actinobacillus mallei, Corynebacterium mallei, Mycobacterium mallei, Pseudomonas mallei, and currently as Burkholdria mallei. The genus Burkholderia was created to accommodate former rRNA group II (Pseudomallei group) Pseudomonas bacteria. It was based on the 16S rRNA sequences, DNA-DNA homology values, cellular lipid and fatty acid composition.

History of rickettsial infections went back to 16^th century and it has long been associated with famine and war. Epidemic typhus had major impact to decide over the outcome of several wars. The epidemic alone killed or caused great sufferings to over 1,00,000 people during World Wars. However, the causative agent was not determined until the early part of the 20^th century. Rocky Mountain spotted fever (RMSF) was the first detected rickettsial infection in 1896 in Idaho Valley, USA. It was originally known as ‘black measles’ due to its clinical appearance in the form of characteristic spotted rash throughout the body. Howard Taylor Ricketts, an American pathologist for the first time described the causative agent of Rocky Mountain spotted fever and was able to isolate it in the laboratory animals during 1902-1909. He was a dedicated scientist and on several occasions injected himself with the pathogen for experiment. The causative pathogen of Rocky Mountain spotted fever, Rickettsia rickettsii was named after him. Later, the family Rickettsiaceae as well as the order Rickettsiales was named after him. Ernest William Goodpasture, an American pathologist and physician, invented the methods of isolation and culturing the rickettsial pathogens in chicken embryos and fertilized chicken eggs.