**3. Pathogenicity of** *Vibrio*

*Vibrio* is usually reported being related to food-borne diseases and aquaculture diseases. Diseases or even death of aquatic animals caused by *Vibrio* infection have been reported worldwide and are showing an increasing trend [7, 11, 18, 67]. Therefore, Vibriosis has caused significant impacts on human health and the development of aquaculture [68–70]. The pathogenicity of *Vibrio* is determined by multiple virulence factors encoded by its virulence genes [71–74]. *Vibrio* infects and destroys the host through a series of processes, including adhesion, invasion, immune escape, in vivo proliferation and production of toxins [46]. *Vibrio* mainly includes adhesion factor, the capsule and polysaccharide, cytotoxin and other virulence factors [75]. Therefore, infection and pathogenesis of *Vibrio* are not completed by a single virulence factor, but the result of the combined action of multiple virulence factors.

#### **3.1 Adhesion factor**

Adhesion is the prerequisite for pathogenic bacteria to cause disease to the body infection, and it is of great significance in invading the host and effectively exerting the virulence [76]. Adhesion is mainly achieved by adhesion factors that specifically recognize and bind to host cells, the ability of *Vibrio* to adhere to and form biofilm on the surfaces of organisms and non-organisms can enhance the virulence and pathogenicity of *Vibrio* [77]. Moreover, adhesion is strongly related to the biofilm formation ability, movement ability and quorum sensing of bacterial [78].

Adhesion factor is a kind of macromolecular substance that can make pathogenic bacteria adhere to the surface of eukaryotic cells, and it plays an important role in the host infection process of *Vibrio*. *Vibrio* has a variety of adhesin, such as fimbriae, cilia, outer membrane protein (OMP), lipopolysaccharide (LPS), extracellular polysaccharide, etc. Among them, fimbriae and cilia belong to fimbriae adhesin, while OMP, LPS, and extracellular polysaccharides belong to non-pilin adhesin [79]. The exposure of OMP to the bacterial surface is a unique and important component of the outer membrane of Gram-negative bacteria, which plays an important role in maintaining the outer membrane structure, ensuring the transport of substances, and stimulating the body to produce antibodies and cytokines. It has been found that the outer membrane protein is an important pathogenic factor closely related to the process of bacterial adhesion and iron uptake [80]. LPS is a lipopolysaccharide substance located in the outermost layer of the cell wall of Gram-negative bacteria. It is not only the main component of the cell wall of Gramnegative bacteria, but also the material basis for the endotoxin of the virulence factor of Gram-negative pathogens [81]. In *Vibrio* pathogens (such as *V. anguillarum*, *V.*  *Community Change and Pathogenicity of* Vibrio *DOI: http://dx.doi.org/10.5772/intechopen.96515*

*vulnificus, V. cholerae*, *V. mimicus*, *V. parahaemolyticus*, etc.), LPS has been proven to be an important pathogenic factor [82].

Although *Vibrio* has a variety of adhesion factors, previous studies on the adhesion of *Vibrio* generally focused on flagellum, and some researchers proposed that flagellum plays an irreplaceable role in the adhesion process of bacteria-infected host [83]. According to the location of the flagellum, the flagellum can be divided into two types: terminal flagellum and peri-flagellum. Belas investigated the differences in the adsorption characteristics of *Vibrio* with different types of flagellum on chitin, and found that peri-flagellum had a stronger affinity for chitin than terminal flagellum [84]. The adhesion of *V. alginolyticus* to the epidermal mucus of *Sparus macrocephalus* was studied by Bordas, which also proved this viewpoint [85]. In addition, studies have found that pili seems to have a strong correlation with the pathogenicity of *Vibrio*. Wright first found in 1989, most clinical isolates of *V. vulnificus* have pili, while environmental isolates lack pili [86]. Analysis of pili protein gene expression during infection indicated that the pili protein expression of the strong strain was higher than that of the weak strain [87]. Moreover, the mobility of the strain was also one of the main factors affecting the adhesion. Kogure found that under the condition of having flagella at the same time, strains with mobility showed faster and stronger adhesion than the strains without mobility [88].

#### **3.2 Capsular and polysaccharide**

After entering the host, bacteria usually activate the host immune system to cause a series of immune responses to eliminate pathogens [89]. In order to survive and reproduce in the host, bacteria must adopt a series of strategies to improve their viability and virulence in the host as well as their resistance to phagocytosis and antibiotics.

The correlation between the capsular and polysaccharide and virulence has been confirmed [90]. The capsule encapsulated on the surface of bacteria is a dense, high molecular weight capsule that plays a major role in evading the host's immune defense. The encapsulated pathogen shows strong resistance to phagocytosis and complement-mediated lethality. Studies have shown that organisms with capsular polysaccharides are more likely to survive in serum, that isolates expressing opaque colonies are more resistant to serum than translucent isolates, and there are differences in colony characteristics between seafood isolates and clinical isolates. Clinical isolates were more resistant to serum complement proteins than environmental isolates, and the clinical genotype had a consistent survival advantage when exposed to serum [91–93]. In addition, the formation of biofilm will also promote the adhesion of pathogens to the host, coordinate the quorum sensing between bacteria, and improve the resistance of pathogens to antibiotics, playing a major role in the escape of pathogens from host immunity.

#### **3.3 Cytotoxins**

Cytotoxic is the main killer factor of pathogens in the process of attacking the host. Toxins secreted by *Vibrio* can be divided into endotoxin and exotoxin. Endotoxin is the lipid part of lipopolysaccharide released after cell death, and the exotoxin is secreted out of cells to cause damage to the host. At present, the toxins produced by *Vibrio* have been studied in depth, and the expression of many virulence factors is related to the pathogenicity of *Vibrio*, including: Thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH), *V. vulnificus* cytolytic toxin (VVC), cholera toxin (CT), and zonulaoccludens toxin (Zot).

*V. parahaemolyticus* is one of the main bacterial isolates of food poisoning caused by seafood contamination and is usually associated with outbreaks of foodborne diseases [11]. The thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) encoded by the *tdh* and *trh* genes are considered to be the main virulence factors of *V. parahaemolyticus*. TDH has a variety of biological activities, such as hemolytic activity, cytotoxicity, cardiotoxicity and enterictoxicity. TDH is a perforated toxin, and its toxic mechanism is to create pores with a diameter of 0 ∼ 2 nm on the erythrocyte membrane, among which the larger pores can make the water and ions in the cells flow out of the cell membrane, and changes in these ion fluxes in the intestine are also the main cause of diarrhea [94, 95]. TRH is a thermolabile toxin, which is similar to TDH in immunology, and can also activate chloride channels and cause changes in ion flux [96]. TDH and TRH share approximately 70% homology [97], and these two genes are considered to be the most important virulence markers of *V. parahaemolyticus* and are commonly used for virulence testing of *V. parahaemolyticus* [98].

*V. vulnificus* is a conditionally pathogenic human pathogen, which can cause severe wound infection, acute gastroenteritis and life-threatening septicemia. In susceptible or immunocompromised individuals, the mortality rate exceeds 50% [99]. VVC encoded by *VvhA* gene of *V. vulnificus* is the key virulence factor of *V. vulnificus,* which mainly plays a role through two mechanisms: cytolysis and apoptosis induction [100]. VVC has species specificity in *V. vulnificus*, which is the only exotoxin that can be secreted out of cells, and belongs to cholesterol-dependent cytolysin of pore-forming protein family [101]. The cytotoxic mechanism of cytotoxin is that it combines with non-esterified cholesterol on cell membrane and aggregates on the cell surface, which makes the cell membrane form a channel and leads to the outflow of intracellular potassium ions, which leads to the rupture of colloid permeable cells. The main mechanism of apoptosis induced by cytotoxin is related to mitochondria. Cytotoxin can lead to the production of mitochondrial reactive oxygen species (ROS) in intestinal epithelial cells, and then lead to cell necrosis and apoptosis [102].

*V. cholerae* has caused several epidemics in history. Cholera toxin (CT) is the main pathogenic factor of O1/O139 *V. cholerae*, which can cause serious damage to intestinal cell function and lead to cholera watery secretory diarrhea [103]. CT is encoded by *ctxA* and *ctxB*. These genes are encoded by CTXΦ of lysogenic filamentous phage and can be transferred between virulent and non-virulent strains [104]. *zot* is also encoded by lysogenic filamentous bacteriophage CTXΦ [105], and its encoded zona-linked toxin (ZOT) is the second virulence index of *V. cholerae*, which can make mucosal cells adhere together, maintain the tight connection structure of mucosal integrity and increase the permeability of intestinal mucosa [106]. Except as a cytotoxin, Zot seems to be related to the assembly of CTX phage in structure and function. *Zot* gene has sequence homology with the coat protein gene, which is probably the coat protein of CTX Φ. This indicates that it may have dual functions [107].

#### **3.4 Other virulence factors**

In addition to secreting toxins, some pathogenic bacteria can secrete a variety of extracellular products, which are also the main factors causing host diseases. For example, Balebona and Morinigo discovered in 1995 that the extracellular products of *V. alginolyticu*s have various enzyme activities such as caseinase, gelatinase, amylase, phospholipase, collagenase, etc., and these extracellular products have strong toxicity to fish cells, which can dissolve fish cells and cause fish death [108]. Balebona et al. infected fish by intramuscular injection with extracellular protease.

*Community Change and Pathogenicity of* Vibrio *DOI: http://dx.doi.org/10.5772/intechopen.96515*

After 6 h, it was observed that the injected extracellular products were lysed, which could lead to fish death in 24 ~ 72 h [109]. Lee et al. found that alkaline serine protease produced by *V.* alginolyticus can reduce thrombin in prawn plasma and prevent hemolymph from agglutinating, which is one of the main lethal factors secreted by *V. alginolyticus. V. vulnificus* metalloproteinases (VVP) is a kind of zinc ion-dependent protease with hemolysis. Miyoshi et al. studies have proved that VVP can enhance vascular permeability, destroy the basement membrane, cause bleeding reaction, cause cell and tissue damage, and eventually develop into sepsis [110].

Iron, as an indispensable trace element, is also an important component of various cellular enzymes, and plays an important role in the growth, reproduction, pathogenicity and cellular metabolism of pathogenic bacteria [111]. The iron carrier of *Vibrio* is an important pathogenic factor, and the iron uptake system mediated by it plays a very important role in bacterial growth and host colonization. Under the condition of lack of iron, the strain will produce a chelation agent iron carrier with high affinity for heme iron ions and low molecular weight, and the iron absorbed from transferrin and lactoferrin will be transported to bacterial cells for its own use through the receptor [112].


**3. Results**

558 A Bird's-Eye View of Veterinary Medicine

The virus may be mechanically transmited by insect vectors such as mosquitoes, mites or ticks, by direct contact with another infected bird, by contact with contamined food, water,

The curse of the disease is influencied by strain, route of infection, and the species of bird

The incubation period of Avipoxvirus is usually less than a week but may be up to 30 days.

Transmission electron microscopy is a classic tool for the diagnosis of poxviruses, where the viral particles with characteristic morphology are present in large numbers in swabs, biopsies or dry crusts (OIE, 2008). This method has typically been used by national reference

The aim of this study was to detect the presence of avianpoxvírus particles in samples of skin lesions of *Gnorimopsar chopi* using negative staining (rapid preparation) immunocytochemistry (immunolabeling with colloidal gold particles) and resin embedding

In the period of the 2005 to 2007, during illegal commercialization of Brazilian birds, 60 baywinged cowbird (*Gnorimopsar chopi*) were apprehended and being forwarded to the CRAS (Wild Animals Recovery Center, Ecological Park of Tietê). After one or two months, 45 birds presented cutaneous lesions in the feet. Some of them also had lesions on the beak and wings. The animals also been presented clinical signs of anorexia, emaciation, locomotion difficulties, diarrhea, dehydration and death. The birds were sent to the Electron Microscopy Laboratory of the Biology Institute of São Paulo to investigate viral agents. Scabs and fragments of skin lesions collected of these birds were processed for transmission electron microscopy utilizing negative staining (rapid preparation), resin embedding and

In the negative staining the scabs and fragments of skin lesions were suspended in phosphate buffer 0.1 M, pH 7.0. Drops of the obtained suspension were placed in contact with metallic copper grids with carbon stabilized supporting film of 0.5% collodium in amyl acetate. Next, the grids were drained with filter paper and negatively stained at 2% ammonium molybdate,

Thin slices of scabs and fragments of skin lesions were fixed in 2.5% glutaraldehyde in 0.1M, pH7.0 phosphate buffer and pos-fixed in 1% osmium tetroxide in the same buffer. After dehydration in cetonic series, the fragments were embedded in Spurr resin (González-

The morbidity rate during an outbreak may reach 100% (Ritchie et al., 1994).

or research laboratories to identify avianpoxvirus (Weli et al., 2004).

Non-specific stress factors are associated with viral reactivation (Ritchie et al., 1994).

semen or surfaces (Metz et al., 1985; Ritchie & Carter, 1995).

(Ensley et al., 1978).

techniques.

**2. Material and methods**

**2.1 Description of the outbreak**

immunocytochemistry techniques.

**2.3 Resin embedding technique**

**2.2 Negative staining technique (rapid preparation)**

pH 5.0 (Brenner & Horne, 1959; Hayat & Miller, 1990; Madeley, 1997).

Among the 45 analyzed animals, all of them (100%) presented in feet, and occasionally in beaks and wings, small yellowish-brown proliferative nodules or scabs of different sizes, characteristic of cutaneous form (fig. 1, arrow).

Fig. 1. Nodular and crusted lesions on the feet of *Gnorimopsar chopi* (arrow).

#### **3.1 Negative staining technique (rapid preparation)**

On the transmission electron microscopy by the negative staining technique, two types of poxvirus particles were visualized in all the analyzed samples. the M form, with regular spaced thread-like ridges comprising the exposed surface (fig. 2, big arrow), measuring 280 x 230 nm and the C form or stain-penetrated particle showing the dumbbell-shaped core (fig. 3, arrow) measuring 360 x 330 nm.

#### **3.2 Resin embedding technique**

Using the resin embedding technique (positive staining) were visualized in the ultrathin sections, three types of intracytoplasmic inclusion bodies. The type A or Bollinger body (fig. 4), outlined by membrane, containing in its interior a great number of mature particles (fig. 4,

Detection of Poxvirus Using Transmission Electron

Microscopy Techniques During Outbreak in Bay-Winged Cowbird (*Gnorimopsar chopi*) 561

Fig. 4. Ultrathin sections of the scabs fragments showing type A or Bollinger

intracytoplasmic inclusion bodies, containing mature particles (arrow). Bar: 1040 nm.

Fig. 5. Ultrathin sections of the skin lesions fragments, showing mature particles with inner dumbell shaped core (big arrow), lateral bodies (minor arrow) and external envelope (blue

arrow). Bar: 280 nm.

Fig. 2. Negatively stained avian poxvirus particles showing the M form, with regular spaced thread-like ridges comprising the exposed surface (arrow). Bar: 180 nm.

Fig. 3. Negatively stained avian poxvirus particles showing the C form or stain-penetred particle reveling the dumbell-shaped core (arrow). Bar: 200 nm.

arrow), measuring 200 x 300 nm, revealing the inner dumbbell-shaped core (fig. 5, big arrow), two lateral bodies (fig. 5 minor arrow) and an external envelope (fig. 5, blue arrow). In the type B electron dense inclusions bodies, viral particles budding of dense amorphous material were observed (figs. 6 and 7, arrow). Fibrillar inclusions constituted by groups of fibrils (fig. 8, arrow) were disposed in groups varying from 2 up to 7 and sometimes showed cross striations.

Fig. 2. Negatively stained avian poxvirus particles showing the M form, with regular spaced thread-like ridges comprising the exposed surface (arrow). Bar: 180 nm.

Fig. 3. Negatively stained avian poxvirus particles showing the C form or stain-penetred

arrow), measuring 200 x 300 nm, revealing the inner dumbbell-shaped core (fig. 5, big arrow), two lateral bodies (fig. 5 minor arrow) and an external envelope (fig. 5, blue arrow). In the type B electron dense inclusions bodies, viral particles budding of dense amorphous material were observed (figs. 6 and 7, arrow). Fibrillar inclusions constituted by groups of fibrils (fig. 8, arrow) were disposed in groups varying from 2 up to 7 and sometimes showed

particle reveling the dumbell-shaped core (arrow). Bar: 200 nm.

cross striations.

Fig. 4. Ultrathin sections of the scabs fragments showing type A or Bollinger intracytoplasmic inclusion bodies, containing mature particles (arrow). Bar: 1040 nm.

Fig. 5. Ultrathin sections of the skin lesions fragments, showing mature particles with inner dumbell shaped core (big arrow), lateral bodies (minor arrow) and external envelope (blue arrow). Bar: 280 nm.

Detection of Poxvirus Using Transmission Electron

groups of fibrils or lamelae (arrow). Bar: 320 nm.

1980; Van Riper et al., 2002; Catroxo et al., 2009).

130 nm.

Microscopy Techniques During Outbreak in Bay-Winged Cowbird (*Gnorimopsar chopi*) 563

Fig. 8. Ultrathin sections of the skin fragments revealing fibrillar inclusions constituted by

Fig. 9. In the immunocitoquemistry technique, the antigen-antibody interaction was strongly enhanced by the dense colloidal gold particles (arrow) over the avipoxvirus particles. Bar:

Lesions similar have been described in other birds (Goodpasture & Anderson, 1961; Trapp,

Lesions can interfere wich perching when occur on the feet or legs and frequently lead to

Clinical signs of lack appetite, weight lost, difficulties of locomotion, diarrhea, dehidratation and death were observed in all birds of this study. This has been documented in similar occurrence of avianpox in other avian species (Mc Donald et al., 1981; Docherty et al., 1991;

Ostrowski et al.., 1995; Gerlach et al., 1998; Kreuder et al., 1999; Catroxo et al., 2009).

development of secondary infections (Van Riper III & Forrester, 2007).

Fig. 6. Ultrathin sections of the scabs fragments reveling type B electron dense inclusions bodies with viral particles budding of dense amorphous material (arrow). Fig. 6 - Bar: 600 nm.

Fig. 7. Bar: 430 nm.

#### **3.3 Immunocytochemistry technique**

In the immunocytochemistry technique, the antigen-antibody interaction was strongly enhanced by the dense gold particles over the viruses (Fig. 9, arrow).


**1. Introduction**

442 Ecosystems Biodiversity

Smith, M.F. & Patton, J.L. (1999) Phylogenetic relationships and the radiation of

Thomé, M.T.C, Zamudio, K.R., Giovanelli, J.G.R., Haddad, C.F.B., Baldissera, F.A. Jr &

Vanzolini, P.E. & Williams, E.E. (1970) South american anoles: the geographic differentiation

Vellend, M. & Geber, M.A. (2005). Connections between species diversity and genetic

Vellend, M. (2003). Island Biogeography of genes and species. Am. Nat., 162, 358–365. Willig, M.R., Kaufman, D.M. & Stevens, R.D. (2003) Latitudinal gradients of biodiversity:

*Mammalian Evolution*, 6, 89–128.

diversity. Ecol. Lett., 8, 767–781.

1018–1031

*Zoologia,* 19, 1–298.

*Systematics*, 34, 273–309.

sigmodontine rodents in South America: evidence from cytochrome b. *Journal of*

Alexandrino, J. (2010) Phylogeography of endemic toads and post-Pliocene persistence of the Brazilian Atlantic Forest. *Molecular Phylogenetics and Evolution* ,55,

and evolution of the *Anolis chrysolepis* species group (Sauria: Iguanidae). *Arquivos de*

patterns, process, scale and synthesis. Annual Review of Ecology. *Evolution and*

Betelvine (*Piper betle* L., family Piperaceae) is an important, traditional and an ancient crop of India and is a shade loving, perennial evergreen climber of tropical origin. It is generally known as "Paan" in Hindi in the Indian subcontinent and by different names in the Asiatic region and is a plant of considerable antiquity. It is distributed in several countries in including India as well as other countries of Indochina region - Indonesia, Malaysia, Vietnam, Laos, Kampuchea, Thailand, Myanmar, Singapore and the Far-East (Figure 1), where its cultivation or ethnomedicinal properties are well known. In India, betelvine is widely cultivated in the states of Uttar Pradesh, Bihar, Madhya Pradesh, Northeastern India, Maharashtra, Karnataka, West Bengal, Orissa, Andhra Pradesh, Tamil Nadu, Kerala and Andamans and almost the entire production of betel leaves is consumed fresh as a masticatory.

Betelvines are dioecious and therefore, under controlled hybridization, attempts have been made to cross different landraces and in some of these experiments, viable seed set has also been reported. However, as a crop, propagation is obligately only through vegetative means. Its cultivation in northern India under sub-tropical conditions (Figure 2) has been shown to be a unique case of plant establishment under anthropogenically regulated microclimatic conditions (Kumar 1999). Cultivated betelvine is grown in traditional farming systems many of which are managed exclusively by families or communities. The betelvine growers invariably named their cultivars with local or vernacular names. These cultivated betelvines are therefore, nothing but landraces and it is this description that will be used consistently throughout the manuscript. A survey over several years indicated between 125 to 150 local cultivars (landraces) of betelvines in India.

SPAR Profiles for the Assessment of Genetic Diversity Between

Male and Female Landraces of the Dioecious Betelvine Plant (*Piper betle* L.) 445

planned because there were no prior reports about chromosomal basis of gender distinction in case of betelvines. A better understanding of how gender discrimination takes place in those plants where chromosomal basis of inheritance of sex is undocumented or negligible or non-existent too is important as the study also assumes significance in understanding the resource allocation and metabolism costs attributed to gender discrimination in those plants where the economic importance of plant is defined by a metabolite or a phytochemical that is recovered from a somatic tissue. Such a study in case of a dioecious plant is expected to throw light on whether or not there is a difference in diversity of the two genders and if so

whether such differences are significant in terms of the economic values of the plant.

Fig. 2. The stages during the construction of the unique man-made structures created to regulate the micro-climate for cultivation of the betelvines in the sub-Tropical parts of India. These structures are called as "bareja" and are almost exclusively made out of natural raw materials such as palm thatch and bamboo. The panels labeled A through H depict the various stages. The small black arrows in panels A through C point to the bamboo sticks that are used to support the betelvines. The white larger white arrows in panels B and C

point to the thatch walls of the bareja sides.

Fig. 1. Schematic map showing the regions of the world where betelvine is cultivated or consumed.

Currently more than 200 landraces are cultivated in India and are often named after the localities, villages or towns where they are grown. As a result of this, though the geographical distribution of the betelvines under cultivation is vast, it is possible that the genetic variation may not be so well distributed. However, no systematic study of betelvine genetic diversity had been carried out to our knowledge. Thus the assessment of genetic variation amongst the different landraces became an important objective for which, PCRbased parallel approaches were used. We have shown, in an earlier study with different landraces using RAPD method, a clear distinction between male and female betelvines or between types of landrace groups (Ranade et al. 2002; Verma et al. 2004). A further substantiation of those results as well as an in-depth analysis was carried out using four different primer sets including RAPD primers on a specific subset of landraces clearly distinguished from each other by flowering behavior as either male or female betelvines (Figure 3). On such a set of known male and female betelvines, hereafter referred to as the "gender set" we have assessed genetic diversity using four primer types revealing polymorphic profiles from discrete but widely distributed genomic regions. These primers include (i) arbitrary sequence decamer primers amplifying from several anonymous regions; (ii) SSR primers amplifying several inter-SSR regions; (iii) minisatellite core sequence primers amplifying from miniatellite rich regions and (iv) primers derived from X and Y chromosomes of a known dioecious plant with heteromorphic sex chromosomes, *Silene latifolia* that were expected to specifically amplify from genomic regions homologous to the X or Y chromosomes in case of the male and female betelvines. Such a PCR-based study was

Fig. 1. Schematic map showing the regions of the world where betelvine is cultivated or

Currently more than 200 landraces are cultivated in India and are often named after the localities, villages or towns where they are grown. As a result of this, though the geographical distribution of the betelvines under cultivation is vast, it is possible that the genetic variation may not be so well distributed. However, no systematic study of betelvine genetic diversity had been carried out to our knowledge. Thus the assessment of genetic variation amongst the different landraces became an important objective for which, PCRbased parallel approaches were used. We have shown, in an earlier study with different landraces using RAPD method, a clear distinction between male and female betelvines or between types of landrace groups (Ranade et al. 2002; Verma et al. 2004). A further substantiation of those results as well as an in-depth analysis was carried out using four different primer sets including RAPD primers on a specific subset of landraces clearly distinguished from each other by flowering behavior as either male or female betelvines (Figure 3). On such a set of known male and female betelvines, hereafter referred to as the "gender set" we have assessed genetic diversity using four primer types revealing polymorphic profiles from discrete but widely distributed genomic regions. These primers include (i) arbitrary sequence decamer primers amplifying from several anonymous regions; (ii) SSR primers amplifying several inter-SSR regions; (iii) minisatellite core sequence primers amplifying from miniatellite rich regions and (iv) primers derived from X and Y chromosomes of a known dioecious plant with heteromorphic sex chromosomes, *Silene latifolia* that were expected to specifically amplify from genomic regions homologous to the X or Y chromosomes in case of the male and female betelvines. Such a PCR-based study was

consumed.

planned because there were no prior reports about chromosomal basis of gender distinction in case of betelvines. A better understanding of how gender discrimination takes place in those plants where chromosomal basis of inheritance of sex is undocumented or negligible or non-existent too is important as the study also assumes significance in understanding the resource allocation and metabolism costs attributed to gender discrimination in those plants where the economic importance of plant is defined by a metabolite or a phytochemical that is recovered from a somatic tissue. Such a study in case of a dioecious plant is expected to throw light on whether or not there is a difference in diversity of the two genders and if so whether such differences are significant in terms of the economic values of the plant.

Fig. 2. The stages during the construction of the unique man-made structures created to regulate the micro-climate for cultivation of the betelvines in the sub-Tropical parts of India. These structures are called as "bareja" and are almost exclusively made out of natural raw materials such as palm thatch and bamboo. The panels labeled A through H depict the various stages. The small black arrows in panels A through C point to the bamboo sticks that are used to support the betelvines. The white larger white arrows in panels B and C point to the thatch walls of the bareja sides.

SPAR Profiles for the Assessment of Genetic Diversity Between

**2. Materials and methods**

stored at -70C till further use.

Male and Female Landraces of the Dioecious Betelvine Plant (*Piper betle* L.) 447

*Plant material*: Betelvine landraces were collected from three of the centers of the All India Co-coordinated Research Project (AICRP) on Betelvine at Chinthalapudi (Bapatla) in Andhra Pradesh, Sirugamani (Tiruchirappalli) in Tamil Nadu and Digraj (Sangli) in Maharastra. The landraces of betelvine for which leaf tissue were collected are listed in Table 1. Young leaf tissue was harvested from the vines, washed free of dirt and dust and then quickly mopped dry on blotting sheets. The leaves were de-ribbed and powdered rapidly in liquid nitrogen, and then either the DNA isolation procedures were immediately followed or the powdered tissue was stored at -70C till further use. The leaves of, the *Piper* species outgroup (*Piper hamiltonii*) as well as non-*Piper* outgroup, Mulberry variety 'MI-0129' were collected from NBRI, Lucknow. As in the case of betelvines the young leaf tissue was harvested, washed free of dirt and dust and then quickly mopped dry on blotting sheets. Then the leaves were de-ribbed and powdered rapidly in liquid nitrogen, and then either the DNA isolation procedures were immediately followed or the powdered tissue was

*Isolation of DNA*: Total plant DNA was isolated from the frozen tissue powder according to the method of de Kochko and Hamon (1990) with some modifications as described earlier (Bhattacharya and Ranade, 2001). At least three to five independent DNA preparations were made from leaf tissues collected from each plant. The quantity and quality of DNA samples were estimated by comparing band intensities on agarose gel as well as by fluorometry

*Minisatellite, SSR, SLXY and RAPD Primers*: Four minisatellite core sequence primers and five SSR primers and three SLXY primers were custom synthesized from Bangalore Genei, Bangalore, India. The fifteen RAPD primers were procured from Qiagen Operon Technology Inc., Alameda, CA, USA. The sequences of all these primers as well as the

207F Bangla Nagaram Chinthalapudi (A.P.)a Jan. 2000 211M Kapoori Chillumurru -- Ditto -- Jan. 2000 213M Kapoori Tuni -- Ditto -- Jan. 2000 214M Kapoori Peddachapelli -- Ditto -- Jan. 2000 218M Kapoori Doddipatla -- Ditto -- Jan. 2000 219M Kapoori Chinnachapelli -- Ditto -- Jan. 2000 223F Bangla (U.P.) -- Ditto -- Jan. 2000 226M Kapoori Vuyyur (A.P.) -- Ditto -- Jan. 2000 234F Godi Bangla (Orrisa) -- Ditto -- Jan. 2000 235F Bangla (M.P.) -- Ditto -- Jan. 2000 239F Bangla Ramtek -- Ditto -- Jan. 2000 244F Kali Bangla (Assam) -- Ditto -- Jan. 2000 301M Kapoori Mhaisal Digraj (Maharashtra)b Jul. 2000 304M Kapoori Bolvad -- Ditto -- Jul. 2000 305M Kapoori Viddi -- Ditto -- Jul. 2000 306M Kapoori Karve -- Ditto -- Jul. 2000

**Source Time**

(DyNA Quant 200, Pharmacia) using Hoechst 33258 as the fluorochrome.

annealing temperatures used in PCR with these primers are given in Table 2.

**Sample # Landrace Collection**

Fig. 3. Photos of the female (top panel) and male (bottom panel) betelvines. The solid white arrows (top panel) and the solid black arrow (bottom panel) point to the female and male inflorescences respectively.


**Author details**

**Figure 2.** Framework on the nexus between organizational trust and organizational conflict management.

46 Organizational Conflict

Furthermore, lack of cooperation will significantly lead to disagreement or misunderstanding over task performance which may result in inter-personality clashes or power struggle among the employees and management that may escalate to organizational conflict. However, one outstanding mechanism for neutralizing conflict in an organization is through organizational trust. This is essentially so because organizational trust produces cooperation, openness, transparency, accuracy, clarity of issues, avoidance of disputes, agreeableness and risk taking in an organization. To buttress this assertion, [57, 58] argued that organizational trust transmits to openness and accuracy in communication and employees who trust each other know that their colleagues will provide them accurate, reliable, and complete information. Consequently, employees who trust one another are more willing to incur the risk of

Oludele Mayowa Solaja

Address all correspondence to<EMAIL_ADDRESS>
Department of Sociology, Olabisi Onabanjo University, Ago-Iwoye, Nigeria


**6. Diagnosis**

The diagnosis of atopic dermatitis is based on a constellation of clinical signs and symptoms, physical examination and history. Laboratory tests are unnecessary and skin biopsy has been found to be little value, but they can exclude other diagnosis in adults. Although the majority of AD patients have elevated total serum IgE, up to 30% of these patients have normal total serum IgE and show no allergic sensitization to food or aeroallergens (Schmid-Grendelmeier, et al. 2005). However, monitoring of IgE remains a nonspecific finding of AD.

Normally, diagnosis is confirmed by chronicity of symptoms, pruritis, and age-specific morphology and distribution of symptoms (Ong P, et al. 2008). However, well-defined criteria are important in the diagnosis of AD and diagnostic criteria developed by Hanifin and Rajka are widely accepted (see Table 1). The American Academy of Dermatology has also developed diagnostic criteria for AD (Leung & Hong, 2007). Please see Table 2.

There are many exacerbating factors that are triggers, which can expedite the progress of inflammation. These triggers are found in food allergens, bacteria, viruses, stress and opportunistic pathogens. One of the major triggers is the microorganism *Staphylococcus Aureus* due to the fact that is organism secretes a toxin which can stimulate action of T cells (Ghazvini, et al. 2010). Among viruses, herpes simplex virus has been identified as one of the possible triggers of AD (Ghazvini, et al. 2010). Diagnostic testing is mainly utilized to determine specific allergens that may be causing the condition. These tests include:

Clinical Management of Atopic Dermatitis 255

Pruritis

reactivity Xerosis

Eczema (with typical morphology for age)

Personal or family history of atopy or IgE

Nonspecific dermatitis of the hands or feet

Positive skin test for immediate-type allergy

Atypical vascular response (facial pallor,

Keratosis pillaris, hyperlinearity of palms

Ocular or periorbital changes, including anterior subcapsular cataracts, Dennie-Morgan infraorbital folds, orbital darkening

Perifollicular accentuation, lichenification or

Metabolic and nutritional deficiencies

polyendocrinopathy, enteropathy, X-

Perioral or periauricular lesions

Immunodeficiency syndromes - Wiskott-Aldrich syndrome

syndrome with Omen's - Immune dysregulation,

Chronic or relapsing history

Early age at onset

Cutaneous infections

Elevated serum IgE levels

white dermatographism)

Pityriasis alba Nipple eczema

prurigo lesions

linked

Table 2. Diagnostic Criteria for Atopic Dermatitis by the American Academy of

Table 3. Differential Diagnosis in Atopic Dermatitis (Deshazo, 2009)7. Treatment

**Essential Features**  (must be present)

**Important Features**

**Associated Features**

diagnosis)

(seen in most cases; add support to

(nonspecific; help suggest diagnosis)

Dermatology (Ong P, et al. 2008)

Other dermatosis - Contact dermatitis - Irritant dermatitis - Nummular dermatitis - Seborrheic dermatitis - Psoriasis ichthyosis

Infections

Malignancies

	- Immediate type reactions such as urticaria, erythema and angioedema may occur without worsening the AD symptoms and they may occur within a few minutes after food intake (Ong P, et al. 2008).
	- Pruritis may be noted after ingestion of food, which makes the patient scratch and may trigger AD.
	- Late reactions may occur as 6 to 8 hours after food ingestion (Ong P, et al. 2008).

\*\*At least 3 major and minor features must be present for a diagnosis of atopic dermatitis.

Table 1. Hanifin-Rajka Diagnostic Criteria for Atopic Dermatitis (Ghazvini, et al. 2010)

 Immediate type reactions such as urticaria, erythema and angioedema may occur without worsening the AD symptoms and they may occur within a few minutes

Pruritis may be noted after ingestion of food, which makes the patient scratch and

 Late reactions may occur as 6 to 8 hours after food ingestion (Ong P, et al. 2008). - Serum IgE measurement – Even though a larger number of AD patients have elevated serum IgE levels, approximately 30% have normal levels and do not present with

**Major Features Minor Features**

Chronic or relapsing dermatitis Ichthyosis, palmar hyperlinearity and

and children Nonspecific dermatitis of the hands or feet

keratosis pilaris

Nipple eczema Pityriasis alba

response

darkening

\*\*At least 3 major and minor features must be present for a diagnosis of atopic dermatitis.

Table 1. Hanifin-Rajka Diagnostic Criteria for Atopic Dermatitis (Ghazvini, et al. 2010)

Raised serum immunoglobulin (Ig) E levels

White dermatographism and delayed blanch

Anterior subcapsular cataracts Perifollicular accentuation Course influenced by

environmental/emotional factors

Dennie-Morgan infraorbital folds, orbital

Facial erythema or pallor

skin reactions which can be observed with this test:

allergic sensitization to allergens (Ong P, et al. 2008).

Typical morphology and distribution Cutaneous infections

adults Early age of onset

excluding other differentials of AD (see Table 3).

Pruritis Xerosis

Flexural lichenification of linearity in

Facial or extensor involvement in infants

Personal or family history of atopy (asthma, atopic dermatitits, contact

urticaria)

after food intake (Ong P, et al. 2008).

may trigger AD.

Table 2. Diagnostic Criteria for Atopic Dermatitis by the American Academy of Dermatology (Ong P, et al. 2008)

Table 3. Differential Diagnosis in Atopic Dermatitis (Deshazo, 2009)7. Treatment

Clinical Management of Atopic Dermatitis 257

The use of natural sunlight for the treatment of atopic dermatitis has been shown to be beneficial, but sunburn should be avoided. If sunlight occurs in the presence of high humidity, or heat which triggers sweating, aggravation of symptoms can occur. Ultraviolet (UV) light (UVB, narrowband UVB, and high-intensity UVA) therapy can be useful in adjunct with other treatments options for patients. Topical glucocorticoid therapy, high-dose UVA1 phototherapy, and UVA-UVB phototherapy were compared in patients with atopic dermatitis. This was a randomized, multi-center trial that found significant differences in favor of high-dose UVA1 and fluocortolone therapy were observed (p < 0.0001), as compared with UVA-UVB therapy. At day 10, high-dose UVA1R was superior to fluocortolone (p < 0.002) therapy. (Krutmann, et al. 1998). There are also several small trials that look at specific wavelengths of and equipment for UV therapy. Chemophototherapy, psoralen with UVA (PUVA), has also been shown to be effective, but should be limited to those patients with widespread AD. Comparison studies with PUVA are limited. Short-term adverse events of UV phototherapy are mild and include: erythema, pruritis, and pigmentation. Potential long-term adverse effects include: premature skin aging, and cutaneous malignancies (Leung, et al. 2004). UV phototherapy is proven to be an effective method of therapy, because of this it is usually used as a

Patients with atopic dermatitis are shown to have significant issues with anxiety, anger, and emotional stress (Bender 2002). They usually respond to embarrassment, frustration, anxiety, or other upsetting events with increased pruritis and scratching (Kodama, et al. 1999). Although emotional factors do not cause atopic dermatitis, studies show that psychological techniques, such as stress reduction approaches, behavior modifications, and group counseling sessions may reduce the exacerbation of atopic dermatitis, particularly

Oral antihistamines are usually recommended for pruritis. However, a recent study of 16 trials showed no little evidence associated with the relief of pruritis with sedating or nonsedating antihistamines (Klein & Clark, 1999). These observations, however, do not exclude the possibility that individual patients may benefit from antihistamines. Because pruritis can be worse at night, oral histamines have been beneficial at bedtime for their sedative properties in patients experiencing symptoms, and can be used as a short-term adjuvant to topical therapy (Kristal & Klein, 2000). The first generation antihistamines, hydroxyzine, diphenhydramine, and cyproheptadine all have sedative effects, but it is shown hydroxyzine to be more effective than the latter (Denman, 1986; Leung, et al. 1998). Newer generation antihistamines ceterizine, loratadine, and fexofenadine may not be beneficial since they lack the sedating properties of the first generation antihistamines. Topical antihistamines should be avoided to the risk of irritating the skin further (Shelley, et al.

**7.1.4 Phototherapy**

second or third line of treatment.

**7.1.5 Psychological approaches**

**7.2 Pharmacological therapies 7.2.1 Nonprescription therapies**

**<IP_ADDRESS> Antihistamines**

those prone to habitual scratching (Melin, et al. 1986).


**2.2. Beyond product innovation**

change management. Assignments have spanned the public and private sectors, particularly

This paper is informed principally by the team's collective direct experience as consulting

On the topic of innovation, the received wisdom is that around 80% of initiatives fail for one reason or another. What are the reasons? Why is the failure rate so high? Is it inevitably something of a random process for which we have no choice but to tolerate 20% success, or are there more predictable and actionable reasons that organization leaders can do something about?

There has been a huge amount of energy expended around improving the innovation process itself and addressing points such as the importance of a strong customer insight, the clarity of the framing of the "customer job to be done" to inform ideation and design thinking, and the helpful rigor required to frame an innovation as "greenfield" investment proposition to

Our observation is that rather less attention has been given to another significant cause of innovation's low strike rate—the ability of an organization to reshape itself so that it is able to

Amidst all the talk of open innovation, partnering, and outsourcing, there is a concern about the ceding of value and control to third parties, but relatively little talk about how organizations can evolve their core—from the high-performance, dominant model that prevail today and toward what they need to become in the future to retain greater ownership of the value

This chapter explores the challenge of "innovation inside" and proposes a new way of framing a response from leadership. It should be emphasized that the thinking is embryonic and very much an "idea under study" with various pilots currently underway with clients. Therefore,

There was a time when innovation was called new product development (NPD). All that mattered was product performance, with customers making rational choices based upon the attributes and relative performance of a product compared to alternatives. The job of NPD was to create a flow of new products (or more often existing products with new/improved

Most of these new products were borne of the same capabilities and expertise as their predecessors. They were designed by the same teams and produced on the same lines. They were

in the CPG, automotive, and financial services and healthcare sectors.

chain and build in greater responsiveness to customer opportunities.

the author would welcome feedback, challenge, and commentary.

features) that could be demonstrated to be superior and preferable.

**2. The changing nature of innovation**

**2.1. Product innovation**

practitioners and to a lesser extent from academic study.

potential investors.

4 Marketing

effectively execute a new proposition.

Twenty years ago, a car produced by one company could set itself apart from that of a competitor based on a number of physical differences. That is patently no longer the case. Value is found not just in what organizations offer but the experience that surrounds it. What might be called a postindustrial model is emphasizing customer experience as one of the few sustainable sources of competitive differentiation.

How have organizations broken out of the product convergence trap? Some have persevered with product innovation but with ever-diminishing returns. Some have tried to brand their way out of trouble, investing heavily to encourage loyalty and build durable emotional bonds with their customers. Others look beyond product, making the most of new digital technologies to innovate in channel, brand, and customer experience. There has also been a considerable "behind the scene" innovation in process and business model, again enabled by technology. Doblin, an innovation consultancy, estimates that while 80% of effort still goes into product innovation, 80% of returns come from innovation elsewhere in the value chain [2].

Going "beyond product" for innovation does appear to be fertile territory from a customer value perspective, but it brings with it many new executional challenges. How to build or gain access to new capabilities, and how to successfully manage an extended network of partners, suppliers, outsource providers? How to retain sufficient value and control within a more complex network of participants? How to maintain some strategic alignment with core business purpose and values?

A global life insurance business found itself in a position where significant innovation opportunities had been identified in relation to healthcare. Rather than just giving people a lumpsum when they were diagnosed with a serious illness, why not go further and offer support across the entire care pathway, helping select the best qualified doctors and best performing hospitals for treatment, helping organize care and rehabilitation, and even helping them with ongoing healthier lifestyle nudges (that will help to reduce their insurance premiums)? This proposition was highly appealing to customers, particularly in countries with less well-developed healthcare infrastructure and availability.

Most established organizations are centered around the "performance cycle" (**Figure 1**). The performance cycle is what creates value for today. It is where core capabilities and processes reside, with focus on consistency, repeatability, targets, control, and strong governance. If the performance cycle is malfunctioning, resource will be drawn from elsewhere, sometimes

Innovation Inside

7

http://dx.doi.org/10.5772/intechopen.74090

For listed companies that need to report quarterly, leadership can become preoccupied with "feeding the monster," fueling, tweaking, and fettling the performance cycle to deliver ever more. The performance cycle attracts the lion's share of talent, resource, and political influence. There is a separate requirement to ponder the future, to provide stimulation and inspiration, and to understand where future value growth is going to come from. Let us call this the innovation cycle (**Figure 2**). The innovation cycle takes into account trends in technology,

sacrificing innovation.

**Figure 1.** Performance Cycle.

**Figure 2.** Innovation Cycle.

This convergence of financial protection with healthcare management might be an exciting and potentially valuable customer proposition, but how do you go about delivering it when your main competence is in actuarial science and product distribution through an agency model? For what healthcare and wellness services would you be a credible provider?

Insurance and healthcare are different sectors that are regulated separately and based on different capabilities. Therefore, it is tempting to respond to such questions with a start point of" we are in the business of insurance, not healthcare" and to preclude the option of organic organization development in favor of partnerships and alliances.

One could have made the same argument about automotive manufacture and distribution compared to vehicle finance and insurance. Yet, over the past 20–30 years, the major automotive brands have successfully evolved their organizations to include sizeable core banking operations.