eChapters

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.

ABSTRACT

Phospholipase A₂ catalyzes the hydrolysis of glycerophospholipids at the sn-2 position and releases fatty acids and lysophospholipids. When the fatty acid is the arachidonic acid, a complementary metabolism leads to pro-inflammatory mediators such as prostaglandins, leukotrienes, thromboxanes and platelet activating factors. Thus, modulating pro-inflammatory lipid mediator production by inhibiting PLA₂ activity remains a potential target for the development of new drugs for the treatment of inflammatory diseases. Medicinal plants have been explored therapeutically in traditional medicines and are a valuable source for drug discovery. Molecular docking studies were carried out for anti-inflammatory compounds from various plant sources with human non-pancreatic secretory phospholipase A₂.

Although in vitro rumen studies of digestibility determination simulates rumen fermentation to some extent, it is almost impossible to incorporate all dynamics of rumen environment in these techniques. Therefore, the in situ Dacron bag technique was developed as an alternative to the in vitro rumen studies (Ørskov and McDonald, 1979). This technique involves placement of feedstuffs in bags made of indigestible fabrics such as nylon, Dacron or silk, directly in the rumen. The loss of dry matter or of any feed component after specific incubation period are measured gravimetrically to determine the rate and extent of degradation.

This technique is widely used, but the procedure differ among the laboratories.Improved methods utilise bags of specific pore size and control the ratio of sample weight to surface area of the bag.Bags with a large ratio of surface area to sample size minimise the error.Optimum pore size is about 40 microns. Smaller pore size retards the entry of organisms and thus inhibit optimum fermentation while larger ones permit transit of lignified particles.

Abstract

Quercus species of family Fagaceae are very important from both economic and ecological perspectives. These exhibit irregular fruiting, and produce seeds recalcitrant to storage, thereby difficult to propagate through conventional procedures. Again because of their very difficult to root nature, extremely limited success has been observed in vegetative propagation. Application of new technological approaches like in vitro propagation may be of interest for accelerating tree improvement programs to these hardwood species. This review provides a summary of the advancement in the application of in vitro propagation to oak species of Kumaun West Himalaya India.

A. In vitro Rumen Fermentation Technique for Forage Quality Evaluation

The term in vitro means “in a glass” in contrast to in vivo which is defined as “within the living body”. In vitro implies a close relationship to an in vivo measurement an in vitro reaction is used as a substitute for a reaction which normally occurs and is observed within a living body. the terms  “artificial rumen” and “in vitro rumen fermentation” are often used interchangeably.

The main advantage of the in vitro techniques rests in the ability to use them to study activity of micro-organisms away form the control and influence imposed by the host animal.

ABSTRACT

Advances in biotechnology, particularly methods for culturing plant cells and tissues, can provide new means for the commercial processing of even rare plants and the chemicals they produce. These new technologies will extend and enhance the usefulness of plants as renewable resources of valuable chemicals. Aristolochic acid is mainly found in the genus Aristolochia. Aristolochic acid has been shown to have anti-tumour activity and has been recommended for its activity against various effects of snakebite and its capacity to inhibit phospholipase A₂ or other enzymes. Considering the importance of medicinal use of A. bracteolata and A. indica, in the present study, they were selected for in vitro development and rapid multiplication of plant through tissue culture. The study was undertaken to enhance the in vitro production of Aristolochic acid in different tissue cultures. Cell suspension culture was established from friable calli of both the species in MS liquid medium. Bergmann plating technique was carried out for cell line selection for more production of Aristolochic acid. Primary transformation was carried out by Agrobacterium tumefaciens strain LBA4404 / pTOK233 with Gus and hpt genes to confirm the transgenic potential of A. bracteolata and A. indica. In order to enhance the production of aristolochic acid in in vitro culture condition, the physical, chemical and biological conditions were altered. Aristolochic acid was isolated, identified and purified from naturally growing plants as well as from tissue culture plants and all other culture staes

1. Introduction

Atalantia monophylla (L.) DC. is a small tree belonging to the family Rutaceae; resembles Citrus in general aspects and is considered a close wild relative of Citrus. The species is commonly called as wild lime, in Malayalam it is called as Kattunaragam and in Tamil Katuuelumichai. A. monophylla is used in Ayurveda, Folk and Siddha systems of medicine. It is used for the treatment of rheumatoid pain and glandular swelling (Sankaranarayan et al., 2010). The root is believed to be antispasmodic (Kiritikar and Basu, 1999), the leaves are often applied for itching and other skin complaints (Panda, 2004) and acridone alkaloids isolated from roots were reported to have anti-allergic activity (Chukaew et al., 2008). The chloroform extract of leaves has shown antiviral activity (Chansakaow et al., 1996). Besides its medicinal value A. monophylla leaves are used as flavoring agent, berries are used for making pickles, juice of berries are used for dyeing purpose and wood as timber. It is also useful as a rootstock for breeding new cultivars of Citrus Linn. (Guha Bakshi et al., 1999). This species has high medicinal value and the natural population is under threat due to habitat destruction, use of wood as firewood, collection of fruits for making pickles and harvesting of leaves, roots for medicinal purposes by the local communities. Hence there is a need for developing an appropriate conservation strategy for this species.

Principle

Orchid seeds are produced in large numbers but are exceedingly small, hence are also called “dust seeds” may be as small as 0.01 by 0.05 mm in dimension and weigh as little as 10-30 ug. Seedling development is generally a slow process in orchids, with the seedlings passing through an initial stage, termed the protocorm stage, prior to the production of distinct vegetative organs (leaves, roots, etc). The protocorms of terrestrial orchids are subterranean and entirely heterotrophic, with all of the nutritional needs of the orchid being supplied by the mycorrhizae (the symbiotic fungus). The length of the protocorm stage is variable, however, in many cases, months, or even years, elapse before leaves and roots are produced.

The combination of a small seed size and fungal symbiont requirement largely precludes the propagation via conventional seed propagation protocols. Accordingly, most of the early research on the development of orchid seed germination protocols focused on in vitro germination with the seeds and fungal symbiont being sown on the same petri plate. Successful protocols were developed for several species; however, the process was complicated by the diversity of fungal symbionts required (across orchid taxa) and the difficulty in isolating and culturing these fungi.

In the 1920’s the process of orchid seed propagation was revolutionized by Lewis Knudson with the demonstration that nutrient media that could be used as a substitute for fungal infection. The isolation and culture of multiple fungal strains was no longer required, and improvements in germination frequency, germination speed, and seedling vigor were generally observed. To date, successful asymbiotic germination techniques, termed ‘flasking’, have been developed for many epiphytic orchids, with several species being saved from extinction via the application of these techniques.

The knowledge and principles from interconnected disciplines of bioengineering, material science and life sciences are combined in tissue engineering with the aim of developing a construct that can wholly or partly restore/maintain/augment the function of a damaged tissue or organ. Tissue engineering construct generally comprises cells on a suitable scaffold. The ultimate functionality of a tissue engineering construct is mainly dependent on the cell behavior on scaffolds. The proliferation and cell viability on scaffolds varies from one cell type to another and from one species to another. So it is important to investigate individual cell-scaffold combinations.

The similarity in structure and composition to bone mineral, osteoconductive properties, ability to integrate with the bony tissue and absence of immune response makes hydroxyapatite [Ca10 (PO4)6(OH)2] the choice of calcium phosphate biomaterial with only disadvantage of slow rate of resorption. On contrary, silica–calcium phosphate composite in comparison to calcium phosphate is rich biomaterials having a faster resorption rate. Studies have shown that coating of hydroxyapatite with a calcium silicate containing layer encourages cell proliferation and osteogenic differentiation of human bone marrow-derived stromal cells.

In this chapter a composite scaffold (calcium silicate, hydroxyapatite and tricalcium phosphate), namely HASi with elements in the following percentages: 66.36 % - calcium, 25.35 % - phosphorus and silicon - 8.29 % and porosity of 50 – 500 µm is undertaken to evaluate the rBMSC proliferation and differentiation properties in vitro with the aim of assessing cytocompatibility of HASi and rBMSC in terms of cell attachment, cell morphology, cell proliferation and osteogenic differentiation.

The estimation of digestibility by feeding trials with sheep or other ruminants is laborious and requires large quantities of feed. Therefore, the chemical composition of feedstuffs are used to predict digestibility by making use of established mathematical relationship between digestibility and composition. However, since the relationship between digestibility and chemical composition varies with the type of feedstuffs, for each group of feedstuffs, mathematical equations will have to be developed by relating chemical composition with in vivo digestibility. In spite of this, the errors associated with this approach of predicting digestibility is large. This has led to the development of techniques that would mimic digestive function of the ruminant in the laboratory.

In vitro methods of feed evaluation using rumen fluid and/or digestive enzymes are the most widely used techniques in ruminant feed evaluation.Although,the technique adopted in different laboratories differ, the principle of the technique is essentially a partially simulated rumen environment in vitro by incubating rumen fluid anaerobically in buffered medium at 39°C for variable period of time. Currently the most commonly used rumen in vitro techniques are,

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.