1.
Biostatistical Methods
by Looney,Stephen W.
Edition: 1stMaterial type: Publisher: India: Humna Press 2002Availability: Items available for loan: UVAS Library [Call number: 570.15195 Looney 1st 2009 29207 Statistics ] (3).
2.
Parasitic Nematodes
by Kennedy, Malcolm | Kennedy, Malcolm W | Harnett, William.
Edition: 2nd ed.Material type: Publisher: UK: CABI; 2013Availability: No items available Checked out (1).
3.
Introduction to Genetic Analysis
by Griffiths, Anthony J.F | Wessler, Susan R | Lewontin, Richard C | Gelbart, William M | Suzuki, David T | Miller, Jeffrey H.
Edition: 8th ed.Material type: ; Literary form:
not fiction
Publisher: USA : W. H. Freeman, 2004Availability: Items available for loan: UVAS Library [Call number: 576.5 Griffiths 17101 8th 2003 Genetics] (1).
4.
Bios Instant Notes in Molecular Biology
by Turner, Phil | McLennan, Alexander | Bates, Andy | White, Michael.
Edition: 3rd ed.Material type: Publisher: [India: Taylor & Francis, 2005Availability: Items available for loan: UVAS Library [Call number: 572.8 Turner 25050 3rd 2005 Genetics] (1).
5.
Molecular Cell Biology / 4th ed
by Lodish, Harvey | Berk, Arnold | Zipursky, Lawrence | Matsudaira, Paul | Baltimore, David | Darnell, James.
Edition: 4th ed.Material type: ; Literary form:
not fiction
Publisher: USA : W. H. Freeman, 2002Availability: Items available for loan: UVAS Library [Call number: 571.6 Lodish 15767 4th 2002 Genetics] (1).
6.
Molecular Biomethods Handbook
by Rapley, Ralph | Rapley, Ralph | Walker, John M.
Edition: 1st ed.Material type: Publisher: New Jersey: Humana Press, 1998Availability: Items available for loan: UVAS Library [Call number: 572.8 Rapley 15568 1st 1998 Genetics] (1).
7.
Essentials of Molecular Biology / 4th ed
by Malacinski, George.
Edition: 4th ed.Material type: ; Literary form:
not fiction
Publisher: [USA] : Jones and Bartlett Publishers, Inc., 2003Availability: Items available for loan: Pattoki Library [Call number: 572.8 Malacinski 15753 4th 2003 Genetics] (1), UVAS Library [Call number: 572.8 Malacinski 17746 4th 2003 Genetics] (3).
8.
Handbook of Biochemistry and Molecular Biology / 4th ed
by Lundblad,Roger L | Lundblad, Roger | Macdonald, Fio.
Edition: 4th ed.Material type: Publisher: USA : CRC Press, 2010Availability: Items available for loan: UVAS Library [Call number: 573.44 Lundblad 24595 4th 2010 Biochemistry] (1).
9.
Cell Growth and Division : A Practical Approach
by Baserga, Renato.
Material type: Publisher: New York: Oxford University Press, 1989Availability: Items available for loan: UVAS Library [Call number: 571.84 Baserga 12898 1st 1989 Genetics] (1).
10.
Introduction to Biotechnology
by Thieman, William J | Palladino, Michael A.
Material type: Publisher: India: Pearson Education, 2009Availability: Items available for loan: UVAS Library [Call number: 660.6 Thieman 23660 2nd 2009 Genetics] (1). Checked out (1).
11.
The Cell : A Molecular Approach / 5th ed
by Cooper, Geoffrey M | Hausman, Robert E.
Edition: 5th Edition.Material type: Publisher: USA : Sinauer Associates, Inc., 2009Availability: Items available for loan: UVAS Library [Call number: 571.6 Cooper 25090 5th 2009 Genetics] (1).
12.
Principles and Techniques of Biochemistry and Molecular Biology
by Wilson,Keith | Wilson, Keith | Walker, John.
Edition: 7th ed.Material type: Publisher: UK : Cambridge University Press, 2010Availability: Items available for loan: Pattoki Library [Call number: 573.44 Wilson 28993 7th 2012 Biochemistry] (1), UVAS Library [Call number: 573.44 Wilson 28926 7th 2012 Biochemistry] (1).
13.
Cell Biology
by Karp, Gerald.
Edition: 7th ed.Material type: Publisher: Asia: Wiley, 2014Availability: Items available for loan: UVAS Library [Call number: 571.6 Karp 29033 7th 2014 Genetics] (1).
14.
Biochemistry and Molecular Biology
by Elliott, William H | Elliott, Daphne C.
Edition: 2nd ed.Material type: Publisher: UK : Oxford University Press, 2001Availability: Items available for loan: UVAS Library [Call number: 573.44 Elliott 18114 2nd 2002 Biochemistry] (1).
15.
Integrated Genomics : A Discovery-Based Laboratory Course
by Caldwell, Guy A | Williams, Shelli N | Caldwell, Kim A.
Edition: 1st ed.Material type: Publisher: UK: Wiley, 2006Availability: Items available for loan: Pattoki Library [Call number: 572.86078 Caldwell 23659 1st 2006 Genetics] (1).
16.
Molecular Biology and Biotechnology
by Walker, John | Rapley, Ralph | Harbron, Stuart | Bugeja, Virginia C | McMullan, Niall | Tsongalis, Gregory | Marks, James | Chaplin, Martin | Adair, John | Cartwright, Elizabeth | Smales, Mark | Fussenegger, Martin | Wright, David | Kalland, Karl-Henning.
Edition: Fifth Edition,Material type: Publisher: UK: Royal Society of Chemistry, 2009Availability: Items available for loan: UVAS Library [Call number: 572.8 Walker 24109 5th 2009 Genetics] (1).
17.
Molecular Biotechnology
by Glick, Bernard.
Edition: 3rd ed.Material type: Publisher: [India] : Unknown, 2003Availability: Items available for loan: UVAS Library [Call number: 660.65 Glick 17112 3rd 2003 Genetics] (1).
18.
Genetic Engineering
by Nicholl.
Material type: Publisher: [UK] : Cambridge University Press, 1994Availability: Items available for loan: UVAS Library [Call number: 660.65 Nicholl 16505 1st 1994 Genetics] (1).
19.
An Introduction to Genetic Engineering
by Nicholl, Desmond S.T.
Edition: 3rd ed.Material type: Publisher: India: Cambridge Universtiy, 2010Availability: No items available Checked out (1).
20.
Modern Genetic Analysis : Integrating Genes and Genomes
by Griffiths, Anthony J.F | Gelbart, William M | Lewontin, Richard C | Miller, Jeffrey H.
Edition: Second Edition.Material type: Publisher: New York: W. H. Freeman, 2002Availability: Items available for loan: Pattoki Library [Call number: 572.8 Griffiths 16512 2nd 2002 Genetics] (1).
21.
Elsevier's Integrated Biochemistry
by PhD, John W. Pelley.
Edition: 1 Pap/Psc ed.Material type: Publisher: China: Mosby, 2007Availability: Items available for loan: UVAS Library [Call number: 616.07 Pelley 23008 1st 2007 Biochemistry] (1).
22.
Practical Skills in Biomolecular Sciences
by Reed,Rob | Holmes,David | Weyers,Jonathan | Jones,Allan.
Edition: 1st ed.Material type: Publisher: Hong Kong: Prentice Hall, 1998Availability: Items available for loan: UVAS Library [Call number: 572.8 Reed 14416 1st 1998 Biochemistry] (1).
23.
Guide to Yeast Genetics and Molecular Biology : Vol.194
by Guthrie, Christine | Abelson, John N | Simon, Melvin I | Guthrie, Christine | Fink, Gerald R.
Material type: Publisher: USA: Academic Press, 2004Availability: Items available for loan: UVAS Library [Call number: 572.7 Guthrie 20831 Vol.194 2004 Genetics] (1).
24.
Recombinant DNA and Biotechnology : A Guide for Teachers
by Kreuzer, Helen | Massey, Adrianne.
Material type: Publisher: USA : ASM Press; 2001Availability: Items available for loan: UVAS Library [Call number: 576.5 Kreuzer 19990 2nd 2001 Genetics] (1).
25.
A Textbook of Genetics and Molecular Biology
by Roychoudhuri, Sabyasachi.
Edition: 1st ed.Material type: Publisher: Kolkata: New Central Book Agency, 2012Availability: Items available for loan: UVAS Library [Call number: 576.5 Roychoudhuri 28907 1st 2012 Genetics] (1).
26.
Molecular and Cell Biology : Based on Schaum's Outline of Theory and Problems of Molecular and Cell Biology
by Stansfield, William D. Cano, Raúl J. Colomé, Jaime S | Cullen, Katherine E (Editor).
Edition: 1stMaterial type: Publisher: Delhi: Tata McGraw Hill; 2010Availability: Items available for loan: UVAS Library [Call number: 572.8 Stansfield 24803 1st 2010 Genetics] (1).
27.
Introduction to Bioinformatics
by T.K.Atwood | D.J. Parry Smith | Samiron Phukan.
Edition: 1st Material type: Publisher: Delhi: PEARSON EDUCATION; 2007Availability: Items available for loan: UVAS Library [Call number: 572.80285 Atwood 28921 1st 2013] (1).
28.
Laboratory Investigations in Cell and Molecular Biology
by Bregman, Allyn.
Edition: 4th Material type: Publisher: U.S.A: Wiley; 2001Availability: Items available for loan: UVAS Library [Call number: 574.87078 Bregman 15337 4th 2002 Genetics] (1).
29.
Guide to Yeast Genetics and Molecular and Cell Biology/ Vol.350
by Guthrie, Christine: Fink, Gerald R | Meurant, Gerard.
Material type: Publisher: U.S.A: Academic Press; 2002Availability: Items available for loan: UVAS Library [Call number: 612.01 Guthrie 20832 Vol.B 2002 Genetics] (1).
30.
Molecular Biology
by David P. Clark.
Edition: 1stMaterial type: Publisher: U.K. Elsevier Academic Press; 2005Availability: Items available for loan: UVAS Library [Call number: 572.8 Clark 24305 1st 2005 Biology] (3).
31.
Short Protocols in Molecular Biology
by Ausubel, Frederick M | Brent, Roger | Kingston, Robert E | Moore, David D | Seidman, J. G | Smith, John A | Struhl, Kevin.
Edition: 5th Edition, 2 Volume Set.Material type: Publisher: U.S.A: Wiley; 2002Availability: Items available for loan: Pattoki Library [Call number: 572.8028 Frederick 17316 5th.Vol.1 2002 Genetics] (1).
32.
Short Protocols in Molecular Biology
by Ausubel, Frederick M | Brent, Roger | Kingston, Robert E | Moore, David D | Seidman, J. G | Smith, John A | Struhl, Kevin.
Edition: 5th Edition, 2 Volume Set.Material type: Publisher: U.S.A: Wiley; 2002Availability: Items available for loan: UVAS Library [Call number: 572.8028 Frederick 17317 5th.Vol.2 2002 Genetics] (1).
33.
Molecular Cell Biology / 4th ed
by Berk, Arnold | Zipursky, S. Lawrence | Matsudaira, Paul | David, Baltimore | Darnell, James | Lodish, Harvey F.
Edition: 4th Material type: ; Literary form:
not fiction
Publisher: U.S.A: W H Freeman & Co; 1999Availability: Items available for loan: UVAS Library [Call number: 571.6 Arnold 24312 4th 1999 Genetics] (2).
34.
Cell and Molecular Biology / 8th ed
by E.D.P. De Robertis.
Edition: 8thMaterial type: ; Literary form:
not fiction
Publisher: Delhi: Wolters Kluwer; 2011Availability: Items available for loan: UVAS Library [Call number: 574.87 Robertis 28988 8th 2011 Genetics] (3).
35.
Molecular Biology and Biotechnology
by Walker, John. M | Rapley, Ralph.
Edition: 5thMaterial type: Publisher: U.K: Royal Society of Chemistry; 2009Availability: Items available for loan: UVAS Library [Call number: 574.88 Walker 23666 5th 2009 Biotechnology] (1).
36.
Molecular Biology
by Weaver, Robert F.
Material type: Publisher: USA: McGraw Hill Higher Education; 2004Availability: Items available for loan: Pattoki Library [Call number: 572.8 Robert 18654 3rd 2005 Genetics] (1), UVAS Library [Call number: 572.8 Robert 19617 3rd 2005 Genetics] (9). Checked out (1).
37.
Handbook of Molecular and Cellular Methods in Biology and Medicine
by Cseke, Leland J | Kaufman, Peter B | Podila, Gopi K | Tsai, Chung-Jui.
Edition: 2nd Material type: Publisher: USA: CRC Press; 2003Availability: Items available for loan: UVAS Library [Call number: 572.8 Cseke 19986 2nd 2004 Genetics] (1).
38.
Essentials of Molecular Biology
by Malathi, V.
Edition: 1sted.Material type: ; Format:
electronic
Publisher: India: Pearson education; 2013Availability: Items available for loan: UVAS Library [Call number: 572.8 Malathi 28916 1st 2013 Genetics] (1).
39.
Laboratory Techniques in Biochemistry and Molecular Biology
by Work, T.S.
Edition: 1st ed.Material type: Publisher: Netherlands: north holand; 1969Availability: Items available for loan: UVAS Library [Call number: 573.44 Work 9786 1st 1969 Biochemistry] (1).
40.
Molecular Cell Biology / 7th ed
by Lodish, Harvey F.
Edition: 7th revised international ed.Material type: ; Literary form:
not fiction
Publisher: New York: W.H. Freeman & Company, 2013Availability: Items available for loan: Pattoki Library [Call number: 571.6 Lodish 30373 7th 2013 Genetics] (1), UVAS Library [Call number: 571.6 Lodish 27496 7th 2013 Genetics] (1).
41.
Molecular Nutrition
by Zempleni, J | Daniel, H.
Edition: 1st ed.Material type: Publisher: UK : CABI; 2003Availability: Items available for loan: Pattoki Library [Call number: 571.6 Zempleni 16475 1st 2003 Genetics] (1), UVAS Library [Call number: 571.6 Zempleni 24117 1st 2003 Genetics] (1).
42.
Phylogenetic Analysis Of Major Fresh Water Carps Of Pakistan Through DNA Barcoding
by Madeeha Awan (2012-VA-650) | Dr. Ali Raza Awan | Dr.Sehrish Firyal | Dr. Muhammad Tayyab.
Material type: ; Literary form:
not fiction
Publisher: 2014Dissertation note: Pakistan is bestowed with the land of geological and topographic diversity. The ecological variation is uniformly reflected in all water lands of the country. Pakistan has significantly huge natural inland water resources in the form of ocean, rivers, networks of canals and lakes (Mirza and Rafique 1994). The country is blessed with one of the largest freshwater resources in the world correspondingly large number of freshwater living vertebrates is available from which fishes are quite significant considering the ecological balance and its consumption as food. It is one of the food sources which solely provide all the essential nutrients, minerals and high quality protein which is not common from other food items (Muhammad Rafique 2007). Out of 33,100 fish species identified worldwide as per Fish Base organization report published in April 2015 (http://www.fishbase.org). Out of 233 (indigenous and exotic) freshwater fish species, 78 economically important indigenous fish species are available in the water bodies of the Pakistan. According to this report fishes are the largest vertebrate group, constituting about 50% of all vertebrate species. Systematically fishes are widely spread in nature, ranging from prehistoric jawless fishes to cartilaginous fishes and also from old to current day bony fishes. The taxonomic placement of these fishes shows their belonging to class Actinopterygii, sub-class Teleostei, 3 cohorts, 6 superorders, 13 orders, 30 families and 86 genera (Rafique 2007; Rafique and Khan 2012).
Demand of fish is increasing day by day not only being the naturally available source of food rather the health benefits associated with its consumption. This necessitates to develop a more efficient and sustainable system to increase their growth. DNA based technologies are being competently employed in aquaculture production fields for pedigree information.
Introduction
2
Moreover, tagging each fish individually is not an easy task so these DNA based methods help in avoiding intrusion of environmental factors which may result from raising fish families in separate reservoirs (Martinez 2007). Fish identification has been traditionally based on phenotypic features. However, due to high multiplicity and morphological similarity, in many cases, fish at its different developmental stages are hard to be identified by relying only on morphological characteristics (Victor et al. 2009).
For phylogenetic studies of the animals the use of mt-DNA is very common and reliable compared to nuclear DNA due to its high evaluation capabilities, which results in gathering of differences even between closely related species (Moore 1995; Mindell et al. 1997).“Bar-coding gap" is the name given to the property that is inter-specific variation in this region is markedly higher than intra-specific variability (Hebert et al. 2003).
Approximately each and every animal contain 13 protein-coding genes (PCGs) as an essential component of their mt-genome (mitochondrial genome), which helps in encoding of several proteins responsible for the oxidative phosphorylation machinery (Richly A et al. 2004, Song H et al. 2008). Being maternally inherited, mt-DNA is better as compared to genomic DNA such as quick evolution, less exposure to recombination, high copy number, high conservation, little duplications and negligible intergenic regions (Waugh J 2007, Xu J 2005). Clonal inheritance is the main property which makes it more worthy and suitable marker in comparison with other available molecular bio-diversity tools (Galtier et al. 2009).
DNA barcoding is one of the taxonomic tools. It is being used to distinguish animal species based on the small segment of their genome such as mitochondrial DNA, designated as an identification tag or barcode of particular species (Herbert et al. 2003). Identification of species using DNA barcoding is quite debatable. Still many researchers consider it as a reliable
Introduction
3
basic tool to ascertain the genetic characterization of diverse eukaryotic species, especially after establishment of the Consortium for the Barcode of Life (CBOL) in 2004 [http: //www. barcodeoflife.org/].
Ideally DNA barcoding should provide quick, reliable and cost effective species identification, even to those user who has little or negligible knowledge of taxonomy (Herbert et al. 2003, Hajibabaei M et al. 2005, Herbert et al. 2005). Identification of unknown source is possible by using distance based tree which can be created by comparing unidentified sequences against retrieved known sequences of different species (Hebert et al. 2003, 2004a, 2004b). DNA barcoding identification system has been recognized universally as standardized method to recognize species and unveil their genetic diversity (Herbert et al. 2003; Herbert et al. 2004). The ideal DNA barcoding is robust, with conserved, universal primer binding sites, reliable DNA amplification and sequencing.
From whole mitochondrial genome, Cytb (Cytochrome b) is considered as one of the most promising gene due to its function and structure, even it is composed of both conserved and rapidly evolving regions which are more related to evolutionary studies (Farias et al. 2001). To identify unknown or ambiguous species it is considered more reliable as it contain sequences which provide the specific information about particular species (Parson W et al. 2000a, 2000b). It is also one of the most useful genetic marker to identify the linkage within families and genera (Meyer 1994; Teletchea 2009). Cytb gene is involved in comparative studies which results in development of new classification schemes and been used to assign a genus to a newly described species as well as improve the understanding of evolutionary relationships of genra (Castresana 2001).
Introduction
4
One of the core objectives of this study is to identify and classify four freshwater indigenous fish species of Pakistan, which includes Labeo rohita (Rohu), Labeo calbasu (Calbans), Catla catla (Thalla) and Cirrhinus mrigala (Mori) using Cytb gene. Morphologically, Labeo rohita shows compressed body with convex dorsal profile while mouth bears a pair of barbells and fins are gray and orange in color. Catla catla shows compressed body with broad head. Mouth is wide with thick lower lip. Labeo Calbasu`s dorsal profile is more convex than that of abdomen and two pairs of barbells are present on fringed upper lip. Cirrhinus mrigala has elongated and streamlined body shape which is grayish and silver in color (Bhuiyan AL 1964; Rahman AK 1989). All of these species are found in freshwater bodies mostly lakes, rivers and ponds except Labeo calbasu which is a bottom dweller. These fishes are harvested by using rod and line or by using nets (Talwar PK and Jhingran AG 1991). These fishes are known as major carps and economically very important for the country due to their high consumption as food. These fishes are also used for fish farming due to their greater muscle mass thus also possess very high commercial value for fish farming business.
Another objective of this study is to resolve the taxonomic anomalies related to above mentioned species. Selling of fish meat in mislabeled packaging is a serious issue now days. Most commonly Hypophthalmichthys molitrix (silver carp) and Ctenopharyngodon idella (grass carp) are sold under the label of Labeo rohita. DNA barcoding is also helpful in detecting such fraudulent mislabeling.
It would be the first study in Pakistan to genetically characterize commercially important fish species. It would help scientists to know about their phylogenetic and taxonomic status and also assist fish fanciers to genuinely identify their species of interest. Identification of fish species is also important for conservation of biodiversity as it helps in preservation and
Introduction
5
identification of endangered species by generating their barcodes from even minimal evidence available. This study has paved the way for molecular biologists to study taxonomic ambiguities at sub species level using SNP (Single nucleotide polymorphism) based identifying marker. Availability: Items available for loan: UVAS Library [Call number: 2207-T] (1).
43.
Polymorphisms Of Bovine Tumor Necrosis Factor Alpha Gene And Its Association With Mastitis In Sahiwal Cows
by Huma Sattar (2013-VA-03) | Dr. Sehrish Firyal | Dr. Ali Raza Awan | Dr. Muhammad Tayyab.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Mastitis is one of the shocking maladies of milch animals causing high production losses to livestock industry of Pakistan (Kenyanjui et al. 2011). It is an inflammatory condition of udder; represent a major problem in dairy cow management. It is one of the most common and frequent disease of dairy industry. Producers suffer a huge loss due to veterinary treatment costs and necessary culling of the infected animals. It negatively affects the milk production, quality of milk, and farm economics (Fourichon et al. 2005). Increasing the disease resistance among dairy cattle is therefore desirable because without controlling mastitis, the national goals of developing dairy farming on commercial and scientific lines and production of wholesome milk which conforms to the standards of WTO Accord would remain elusive.
Mastitis is inflammation of udder that caused by physiological and metabolical changes (Schalm and Noorlander 1957). There are two main types of mastitis; clinical mastitis (characterized by classical symptoms i.e., swelling of udder, redness, clumps and clots in milk etc) and sub-clinical mastitis (not show any symptoms, Milk appear normal, udder appear normal) (Schrick et al. 2001). Mastitis is ranked as a top disease of dairy herds (Rinaldi et al. 2010). This mammary gland infection caused by pathogenic micro organisms such as Staphylococcus aureus, Streptococcus uberis, and Esherichia coli in the mammary gland (Heringstad et al. 2000).
India, China and United States are the larger producer of milk and Pakistan is on forth number in milk yield. Pakistan almost produces 36.5 million tons of milk yeild per year (Cady et al. 1983).The Sahiwal breed is well known among for its superior dairy qualities (Barker et al. 1998). Both cross and pure breed Sahiwal cows have high milk production rate (Khan et al. 2013).
It is very difficult to comprehend this disease because numerous environmental and genetic factors are involved in the origin and development of mastitis (Bradley 2002; Carvajal et al. 2013). Susceptibility and resistance to mastitis is a complex trait influenced by genetic variation of animals. Among these variations, the polymorphisms in immunity genes are principal key factors in defensive mechanism of mammary gland (Ibeagha-Awemu et al. 2008).
The mammary gland tissue is protected by immune system by two defense system; innate and acquired immunity. Innate immunity response by the host is a quick response of bacterial defense system (Mesquita et al. 2012). Innate system is a rapid and effective mechanism that activated on recognition of antigen (Akira et al. 2006). Innate immune system is activated when specific pattern recognition receptors (PRR) that are present on the surfaces which are attach to the specific pathogen (Shuster et al. 1996). PRR are presnt on leucocytes in milk and on the epithelial cells lining of udder. It is reported that T- lymphocyte subset i.e., CD4+, CD8+ and ɤδT are present in infected bovine mammary glands. (Goldammer et al. 2004; Strandberg et al. 2005).
Innate defense (nonspecific) of the mammary gland is stimulated by the physical barrier such as teat end, natural killer (NK) cells, neutrophils, macrophages and certain other soluble factors. The teat cannals are considering the main line of defense. Microorganisms enter from teat canal in milk. The main roles of teat sphincter muscles are to remain orifice close so that bacteria cannot enter. This teat canal also lined with keratin, whose estrified and non estified fatty acid function as bacteriostatics that provide protection and play role to eliminate bacteria causing mastitis (Oviedo-Boyso et al. 2007).
If a pathogen is not eliminated by the physical barrier, the acquired immune system is triggered. In comparison, this system is much faster than other immune response. The memory response is significantly stronger, long durable and more efficient to kill the pathogen. The acquired immune system (memory response) have ability to differentiate self or nonself cells and produce antibodies only against antigens through membrane bound protein called major histocompatibility complex (MHC) molecules. Specific immune system activate only when antigens bind with an MHC that is present on the surface of certain cells, this process is referred as antigen presentation. Recognition of pathogenic factors for elimination is mediated by macrophages, several lymphoid, and immunoglobulins (Ig) or antibodies (Sordillo and Streicher 2002).
The most acute responding macrophages and T-cell cytokines are TNF-α, LTF, IL1, IL6, IL8, and IFN-ɤ present in intramammary infection in cows. These genes play important role in improvement of immunity to mastitis (Burton and Erskine 2003).
Tumor necrosis factor alpha is main pro-inflammatory adipokine that is part of systematic immune defense. The main function of TNF-α gene is responsible for proliferation, differentiation and activity of many immune system cells; B lymphocytes, NK (natural killer). It also induces the production and release of many other cytokines (Wojdak Maksymiec et al. 2013) and also enhances the chemotactic and phagocytic effects of immune response. TNF-α gene contains four exons and three introns that are present on chromosome BTA23q22 (Bannerman 2009; Moyes et al. 2009).
TNF-α is a member of a group of cytokines that stimulate the specific immune system. TNF consist of 212 amino acid arranged in stable homotrimers (Kriegler et al. 1988; Tang et al. 1996). The 17-kilodalton (kDa) TNF protomers are composed of two β-pleated sheets and β-strands, joined together antiparallel (Tang et al. 1996).
TNF-α is a component of natural protection systems of humans and animals. Milk gives nourishment and disease resistance to the new born. Various cellular and soluble immune components are important for protecting the mammary gland from infectious diseases like mastitis. Mastitis affects one third of all dairy cows and cost the dairy industry about 2 million dollars annually (National Mastitis Council (1996). Dairy cattle are especially susceptible to mastitis due to diminished mammary gland defense mechanisms (Sordillo and Streicher 2002).
TNF-α is not only produced by activation of macrophages, but also other cell types such as CD4+ lymphocytes, NK cells, neutrophils, mast cells, eosinophils, and neurons. Large amounts of TNF are released in response to lipopolysaccharide, other bacterial products, and Interleukin-1 (IL-1).TNF-α stimulates the proliferation, differentiation and activity of many immune system cells; B lymphocytes, NK (natural killer). TNF-α induces the release of many other cytokines (Wojdak-Maksymiec and Mikolajczyk 2012). TNF-α also enhance the chemotactic and phagocytic effects of immune response.
. The present study is designed to determine the genetic polymorphism in exon 4 of TNF-α gene of mastitic cows and its association resistance and susceptibility towards mastitis.
Availability: Items available for loan: UVAS Library [Call number: 2224-T] (1).
44.
Sequence Analysis Of Mitochondrial Atpase 8/6 Gene Variants In Equine
by Kashif Hameed Anjum (2012-VA-905) | Dr. Asif Nadeem | Mr.Maryam Javed | Dr. Muhammad Tayyab.
Material type: ; Literary form:
not fiction
Publisher: 2014Dissertation note: Human has been using horses for doing different jobs like transportation, hunts, carrying loads, warfare and sports (Zhang et al. 2012). In Pakistan, horses and donkeys are mostly used for transportation whilehorses are also used for racing and playing games like polo.There are two main types of horses:Equuscaballusare domesticated horses and Equusferus are the wild horses. There are more than 300 breeds of horses in the world today (Barbara and Dafydd, 2007). The horse population is estimated as 0.32 million and has been decreasing over the years in Pakistan. Main breeds of horses that are found all over the Pakistan are Kajlan, Kakka, Balochi, Morna, Shien, Anmol, Makra, Pak-thoroughbred,Heerzaiand Waziri (Khan, 2004).
Seventy percent of the population earns living from the land. Agriculture contributes nearly 21% to gross domestic product and generates 43% of all jobs. Over 30 million people in rural areas derive their livelihood from livestock production. The number of impoverished communities moving from the country to find work in Pakistan’s towns and cities is rising. Many of these people rely on working equine animals to earn a living.
Nuclear and mitochondrial genomes are frequently used in animal genetic research. Nuclear genomeis generally a huge and complicated molecule and is not well studied in many species. However mitochondrial DNA being small sized and having high mutation rate is used frequently for the purpose of genetic research (Stanley et al. 1994). Characteristic of having fast evolution rate as compared to nuclear DNA makes mitochondrial genes a good tool for genetic studies (Avise, 1994).
Several studies have investigated the genetic relationship among horse and donkey breeds using mitochondrial sequences as a marker for breed characterization and phylogenetic. Each mitochondrion contains its own circular DNA, replication, transcription and translation machinery and serves as semi-autonomous organelle. Mitochondria perform so many important functions in our body like metabolism(oxidative phosphorylation), apoptosis and aging(Weinberg, 2007).
The advent ofpolymerase chain reaction and direct sequencing techniques with the use of mtDNA as a phylogenetic marker has been extended to much greater levels of phylogenetic inclusiveness (Zardoya and Meyer,1996). The special features of mtDNAi-e,lack of introns, maternal inheritance, absence of recombination events and haploidy have made it the most common type of sequence information used to estimate phylogenies among both closely and distantly related texa(Meyer, 1993).
Four of the five mitochondrial respiratory chain complexes, namely C1, C3, C4 and C5 (ATP synthase) contain subunits encoded by mitochondrial DNA (Kadenbach, 2012). ATP synthase (Complex5) functions to make ATP that is used by the cell (Von et al. 2009). ATP synthasecomprisesan integral membrane cylindrical, the F0 particle and a peripheral matrix-facing F1 particle, the catalytic ATP synthase domain (Boyer, 1997). All aerobically respiring organisms possess ATP synthase enzymes and are located inthe cell membrane in prokaryotes, the mitochondrial inner membrane in eukaryotes and the chloroplast thylakoid membrane (Ackerman and Tzagoloff, 2005). This enzyme is responsible for the final step of oxidative phosphorylation. The protons move down their concentration gradient from inter membrane space to matrix through F0 particle while F1particleuses the energy provided by influx of these protons and converts ADP molecule into ATP. ATPase 6 and ATPase 8 proteins are components of F0 particle where they play direct role in maintaining the structure and function of ATP synthase (complex 5). All five subunits of F1 and most of the F0 subunits are nuclear encoded(Collinson et al. 1996). Only two proteins i-e, ATPase 6 and ATPase 8 are encoded by mtDNA (Boyer, 1993).
The present study is designed to investigate the diversity and phylogenetic analysis of Thoroughbred Pakistani horse and donkey breeds on the basis of ATPase 6 and ATPase 8 genes.
Availability: Items available for loan: UVAS Library [Call number: 2236-T] (1).
45.
Dictionary of Microbiology and Molecular Biology / 3rd ed
by Singleton, Paul | Sainsbury, Diana.
Edition: 3rd Edition.Material type: ; Literary form:
not fiction
Publisher: India : Wiley; 2002Availability: Items available for loan: UVAS Library [Call number: 572.803 Singleton 15302 3rd 2002 Microbiology] (1).
46.
Diagnostic Molecular Pathology : A Practical Approach / Vol.1
by McGee, James O'D | Herrington, C. S.
Material type: Publisher: UK: Oxford University Press; 1992Availability: Items available for loan: UVAS Library [Call number: 616.0756 McGee 24877 Vol.1 1992 Pathology] (1).
47.
Diagnostic Molecular Pathology : A Practical Approach / Vol.2
by McGee, James O'D | Herrington, C. S.
Material type: Publisher: UK: Oxford University Press; 1992Availability: Items available for loan: UVAS Library [Call number: 616.0756 McGee 24878 Vol.2 1992 Pathology] (1).
48.
Nutritional Genomics : Impact on Health and Disease
by Brigelius-Flohe, Regina | Brigelius-Floh�, Regina | Joost, Hans-Georg.
Edition: 1st ed.Material type: Publisher: Germany : Wiley-Blackwell; 2006Availability: Items available for loan: UVAS Library [Call number: 515.10724 Brigelius 19632 1st 2006 H.Nutirition] (1).
49.
Expression And Mutational Analysis Of Breast Cancer Susceptibility Gene 1 (Brca1) And Cyclooxygenase-2 (Cox-2) Gene In Feline And Canine Tumours
by Haleema Sadia (2007-VA-567) | Dr. Muhammad Wasim | Prof. Dr. TahirYaqub | Dr. Abu Saeed Hashmi.
Material type: ; Literary form:
not fiction
Publisher: 2014Dissertation note: Cancer is the first cause of death in cats and dogs while in human it is the second most cause of death (Jemal et al. 2008). According to an estimation, cancer related deaths in the world are 13% and 70% of these deaths are in poor countries (World Health Organization 2012). Such natural cases of cancers in cats and dogs especially, in dogs offer an opportunity to use the dogs for comparative cancer studies and as an animal model for anticancer drug development (Pawaiya 2008). Inu a series of more than 2000 autopsies, it was found that almost forty five percent dogs that lived for ten or more years expired because of cancer (Bronson 1982). Dogs are affected by skin cancer 35 times more often than humans. They are also affected 4 times more often by mammary gland cancer, 8 times more often by bone cancer, and twice more often by leukemia, than humans (Cullen et al. 2002). The regulation of cell proliferation, genome stability and programmed cell death are important for systemic homeostasis.
1.1Historical perspective on cancer causation
Hippocratic and Galenic medicine attributed the spread of black bile (one of the four humours) in the tissue as the cause of the cancer (Diamandopolus 1996) is an idea survived intact through the Middle Ages and Renaissance. With the discovery of the lymphatic system by Gasparro Aselli in 1662, the black bile theory was superseded by the idea that cancer was an inflammatory reaction to extravasated lymph; a theory modified 150 years later by John Hunter who introduced the notion that contaminated coagulating lymph was the origin of the cancer (Kenneth 2003). A German pathologist Johannes Muller first time demonstrate that cancer is made up of cells (1838) but he also gave an idea that cancer cells were originated from a bud called Blastema instead of normal cells (Kardinal and Yarbro 1979). Following Schleiden and Schwann's cell theory of tissues,it was Rudolf Virchow (Muller’s student) who in 1855 demonstrated that every cell was derived from another cell (omnis cellula e cellula), including cancer cells (Mazzarello 1999; Porter 1999). In 1867 Wilhelm Waldeyer supported the theory of the normal cell for the origin of cancer and he believed that metastasis resulted from transportation of cancer cells by blood or lymph (Porter 1999).
Around the turn of the twentieth century the beginning of tumour transplantation experiments led to the new view of the cancer cell as an autonomous cell. The first successful tumour transplants were described in 1876 by the Russian veterinarian Mstislav Aleksandrovich Novinski (Novinski 1876). He reported in his thesis entitled “On the Question of the Inoculation of Malignant Neoplasms” the first successful serial passage of tumours through transplantation in dogs. Novinski's transplantation experiments were based on the inoculation of canine transmissible venereal tumour (CTVT) in puppies. Novinski stated that successful tumour transplantation depends on the inoculation of a living element of the tumour and that the transplantation of the element of a cancerous tumour to healthy tissue acts as an infecting agent. In 1888 Wehr repeated Novinski's transplantation experiments in dogs with similar results (Shimkin 1955). It is interesting to note that the dogs used for transplantation of CTVT did not come from a single breed and were therefore not highly inbred. The allo-transplantation of tumours seemed less surprising in the late 19th Century than it does today with our modern knowledge of histo-incompatibility.
The successful results obtained with CTVT served as model for tumour transmission in other animals. Hanau in 1898 inoculated two rats with vulvar epidermoid carcinoma and observed growth of the tumour in the recipients (Shimkin 1955). In 1901 Leo Loeb supported the transplant ability of tumours in rats (Witkowski 1983; Brent 1997). In 1903 a Danish veterinarian Carl O. Jensen determined the successful growth of transplanted tumours in mice by heredity (Brent 1997). The discovery that the tumour could be successfully transplanted into (Witkowski 1983; Brent 1997) other mice, led the scientists to use rodent system to supply tumours for experiments. The observation that a single tumour could be expanded through many generations exceeding the life span of the laboratory mouse led Leo Loeb to the "cancer immortality" concept (Witkowski 1983).
The earliest observations reported by John Hill in 1759 and by Percival Pott in 1775 on the association of a specific tumour to a specific profession or work, led to the idea that some chemicals can cause cancer (Greaves 2000). In 1918 Yamagiwa and Ichikawa induced cancer by applying coal tar to rabbit skin(Greaves 2000; Luch 2005). After the discovery of the X rays by Wilhelm Conrad Roentgen in 1895, Frieben published data in 1902 indicating that cancer rates were increased among persons working with X-rays (Cassileth 1983; Greaves 2000)
1.2 Tumour Progression
The first detailed characterization of the dynamic nature of cancer was described by Leslie Foulds (Foulds 1949). Foulds showed that tumours progress (evolve) through different stages, characterized by the acquisition of different phenotypic traits such as increased growth rate, hormone dependence, invasiveness, formation of metastasis (Foulds 1949; Fould 1954; Foulds 1957). With the progress of molecular biology the phenotypic view had been replaced with the somatic mutation theory, where cancer evolved through the accumulation of different mutations in several genes (Greaves 2000). The accumulation of mutations in somatic cells implicated the presence of different cells bearing different mutations and also the presence of natural selection, which selected the cells with advantageous mutations. One of the questions arising from the somatic mutation theory was whether a tumour had a single or a multiple origin.
This observation was supported by a karyotype analysis in chronic myelogenous leukaemia (CML) by Peter Nowell and David Hungerford in 1960 (Nowell 2002). They described the presence of an unusually short chromosome 22 in all CML tumour cells analyzed, and the absence in the normal cells from the same patients. This observation suggested that this mutation was a somatic mutation that occurred in one cell in the bone marrow, which gave it a selective advantage to expand as a clone. Nowell postulated that a tumour develops by a Darwinian evolutionary process, where cells with mutations conferring a growth advantage are selected and expanded (Nowell 1976; Greaves 2002). In 1954 Peter Armitage and Richard Doll analyzed human cancer incidence over the age, and showed that chances of cancer increased in older people (Armitage and Doll 1954).
The concept that cancer might be contagious also recurs throughout the past 300 years.In the 17th and 18th centuries, physicians Daniel Sennert and Zacutus Lusitanus supported the hypothesis that cancer was contagious. In fact in 1779 a hospital in Paris was directed to move the cancer patients from the city (Cassileth 1983; Kenneth 2003).
1.2.1 Exogenous and endogenous factors
In 1844 the Italian physician Domenico Antonio Rigoni-Stern noted that cancer of the cervix was frequent among married ladies, rare among unmarried ladies and absent in Italians nuns. In contrast, breast cancer was more frequent among nuns (Greaves 2000). These observations led to the hypothesis that cervical cancer was sexually transmitted, and we now know that the cause is a papilloma virus (Hausen 2002).In 1908 Wilhelm E and Olaf B, transferred the leukemia in chicken by tissue filterates (Wyke 2003). In 1911, Peyton Rous demonstrated that viruses were the cause of solid tumours (Sarcoma) in chickens but it took many decades before his data were accepted (Dulbecco 1976). The notion that viruses can cause cancer was a discovery that brought back the fear that cancer was a contagious disease.
There are many exogenous and endogenous risk factors that affect the tumor suppressor genes and oncogenes (Todorova 2006). Tumour viruses (Bishop 1980), chemical carcinogens (Loeb et al. 2000), natural chemicals, (Ames et al. 1990), herbicides (Glickman et al. 2004), physical carcinogens like radiation (Upton 1978) are exogenous factors while inherited genetic defects, immune system (Rosenthal 1998) and hormonal factors (Rodney 2001) are among endogenous risk factors.
Although tumour cells are generally described as independent evolving units, recent results suggest that tumour cells are able to stimulate stromal cells to produce growth factors that increase tumour proliferation (heterotypic stimulation) (Kinzler and Vogestein 1998; Skibe and Fuseing 1998; Iyengar et al. 2003). It has been demonstrated that cells involved in the immune response to tumours may produce factors such as inflammatory chemokines that may also promote the tumour proliferation (Pollard 2004; Wyckoff et al. 2004)
1.2.2 Two hit hypothesis
Retinoblastoma is a tumour that becomes manifested early in life. Retinoblastoma can be inherited or sporadic. According to the two hit hypothesis in the inherited form a single mutation in the Retinoblastoma (Rb) gene is present in the germ line which gives the genetic predisposition to develop cancer, but a second mutation in the normal Rb allele which occurs in the retinoblast must be acquired to develop cancer (Knudson 2001). In the sporadic form the two mutations in the Rb alleles occur in the somatic cells. Although the epidemiological and molecular observations have consolidated the multistage theory of cancer, the number of mutations and in which sequential order they have to be acquired to develop cancer is still an open question (Hanahan and Weinberg 2001; Hahn and Weinberg 2002b).
1.2.3 Oncogenes
Early experiments involving transforming retroviruses and the transfer of genes from tumour cells into established rodent cells allowed the identification of several cancer causing genes called oncogenes. The result of these experiments suggested that cancer could be induced by the mutation of one proto-oncogene. However, the rodent cells used as recipient in the gene transfer experiments were not normal, but were immortalized, thus acquiring the ability to proliferate indefinitely. When the normal rodent cells were used, the transfer of a single oncogene failed to induce transformation, while the transfer of two oncogenes resulted in transformation. Human cells require more mutations than rodent cells and that there are differences also between cell types within the same species (Rangarajan et al. 2004).
1.3 Cancer Hallmarks
Despite the enormous variety of tumours affecting different types of tissues in animals and humans, research over the past 50 years has revealed that all malignant cancers share the same essential alterations (Hanahan D and Weinberg RA 2000).
These hallmarks include:
Immortalization
Evasion from programmed cell death (apoptosis)
Independence from growth stimulation
Resistance to growth inhibition
Angiogenesis
Invasion and metastasis
Genetic instability.
These hallmarks are briefly described below.
1.3.1 Immortalization
Telomeres contain DNA sequence repeats and protein. The repeat sequence consists of hexameric motifs such as GGGTTA in humans, extended for 10 –20 kilobases. The 3’ end has a 100-400 nucleotide over-hang (Mathon and LIoyd 2001). Telomeric DNA is generated by an enzyme called Telomerase Reverse Transcriptase (TERT) which has two subunits, RNA and catalytic protein subunit. This RNA binds the telomeres DNA ends thus acting as template for telomere elongation. The chromosome ends are protected by several proteins: TRF-1, TRF-2, and POT–1 (Mathon and LIoyd 2001; Hahn and Weinberg 2002a). Several experiments have shown that senescence is activated when the telomeres are shortened down to 5 kb and that senescence is triggered by the shortest telomere present in the cell (Hemann et al. 2001).
Many reports have suggested that the replicative senescence is not activated by the erosion of the double strand repetitive sequence, but by the degradation of the 3’end single strand overhang, resulting in loss of protective capping (Stewart et al. 2003). Telomere length is maintained by the activation of telomerase or by an alternative mechanism called alternative lengthening of telomeres (ALT), where the telomeres are regenerated through recombination-based inter chromosomal exchange of sequence information (Bryan et al.1997; Dunham et al. 2000). In the normal cell telomerase is transiently expressed, since it can be detected only in S phase, but in neoplastic cells its expression is increased and is detectable throughout the cell cycle (Mathon and Lloyd 2001). In tumour cells the senescence and crisis barriers are avoided by the activation of telomerase regenerating the telomeres and by the inactivation of tumour suppressor and pro-apoptotic genes (Hanahan and Weinberg 2000; Hahn and Weinberg 2000b).
1.3.2 Apoptosis.
The sensors detect the intra- and extra-cellular signals. The intracellular signals include DNA damage, hypoxia and oncogene overexpression (Evan and Littlewood 1998). The extracellular signals monitor the cell-cell and cell-matrix homeostasis (Aoshiba et al. 1997; Prince et al. 2002; Alberts et al. 2002a). The signals detected by the sensor are mainly conveyed to the mitochondria, where a series of cytoplasmatic proteins of the Bcl2 family control the release of cytochrome C from the mitochondria (Alberts et al. 2002a). The release of cytochrome C activates an array of intracellular proteases called caspases causing protein and DNA degradation (Hanahan and Weinberg 2000). The caspases can be directly activated by extracellular proteins such as FAS ligand, which binds to the death receptor FAS (Houston and O’ Connell 2004).
Once the caspase cascade is triggered it cannot be inactivated (Alberts et al. 2002a). It has been reported that the tumour suppressor p53 can trigger the caspase cascade by the overexpression of the Bax protein, a member of the Bcl2 family, which in turn increases cytochrome C release thus inducing apoptosis (Hanahan and Weinberg 2000). In CTVT it is likely that expression of c-myc is up-regulated, due to insertion of a LINE-1 element as discussed later. Ectopic c-mycexpression can promote tumour growth and survival, as seen, for instance, in immunoglobulin gene c-myc chromosome rearrangements in Burkitt's lymphoma (Hemann et al. 2005).
1.3.3. Independence from growth stimulation
1.3.3.1. Growth factors
Thus the proliferation of a cell is dictated by the needs of the cells around it (Hanahan and Weinberg 2000). In contrast, a tumour cell escapes from the external dependence to become an autonomous evolving unit, by producing its own growth signals.
1.3.3.2 Growth factor receptors
Another mechanism selected by tumour cells is the overexpressions of growth factor receptors, which induce the tumour cells to become sensitive to concentrations of growth factor that normally, do not trigger proliferation (Hanahan and Weinberg 2000). Proliferation can also be induced by a mechanism independent of the growth factor, for example the alteration of the cytoplasmic tail of growth factor receptor causes self-activation of the receptor, which therefore becomes independent from the external microenvironment (Alberts et al 2002b).
1.3.4 Resistance of growth inhibition
Like growth signals, the anti-proliferative signals derive from soluble factors or surface proteins that are produced by neighbouring cells, or are induced by components of the extracellular matrix (Hanahan and Weinberg 2000; Alberts et al. 2002d). These external inhibitory signals activate different intracellular pathways that regulate the cell cycle (Alberts et al. 2002c).
The Rb protein and its related proteins, p107 and p130 play a key role in controlling this transition (Weinberg 1995). The association of Rb with the transcription factor E2F inhibits the transcription of genes involved in the G1-S progression (Alberts et al. 2002c). The hyper-phosphorylation of the Rb protein induces the dissociation with E2F, therefore allowing progression to S phase (Alberts et al. 2002c). Normally complexes of cyclin and cyclin dependent kinase (CDK) induce the phosphorylation of the Rb protein (Alberts et al. 2002c). Many tumours can avoid the antigrowth signals by altering Rb activity or the proteins involved in Rb phosphorylation (Mittnacht 2005).
1.3.5 Angiogenesis
Although the majority of the new vessels in adult tissues are derived by sprouting from existing vessels, many evidences indicate that progenitor endothelial cells are derived from the bone morrow contributing to the vessel growth (Zhang et al. 2000; Contreras et al. 2003; Nishimura and Asahara 2005; Religa et al. 2005). Although endothelial cells are highly proliferative in response to several angiogenic factors, they have long half-lives up to several years (Carmeliet 2003). In order to adapt the vascular system to the tissue's requirements, several mechanisms regulate the process of angiogenesis (Carmeliet 2003). A key molecule involved in the angiogenesis process is the vascular endothelial growth factor (VEGF) (Carmeliet 2003). In addition it has been demonstrated that tumours can activate or inactivate pro- and anti-angiogenic factors respectively present in the extracellular matrix by producing several proteases (Gately et al. 1997; Harlozinska 2005).
1.3.6 Metastasis
In cancer during tumour progression, some tumour cells acquire the ability to migrate and form new colonies at secondary sites and these cells then make new tumour cells (Hanahan and Weinberg 2000). It has been estimated that 90 % of mortality associated with cancer is due to metastasis (Sporn 1996). Results show that few cells in the primary tumour acquire the ability to grow in the secondary sites and that the tendency to metastasise is acquired in the early steps of tumour progression (Van’t Veer and Weigelt 2003).
Progressive alteration of normal tissue homeostasis by tumour and stromal cells, allow tumour cells to move throughout degraded matrix, and to invade surrounding tissues (Hanahan and Weinberg 2000). Tumour cells are also aided to migrate by soluble factors (chemotaxis) and bound adhesion molecules (haptotaxis) (Nguyen 2004). In order to invade new organs, circulating tumour cells need to stop and exit the systemic circulation. In an unspecific manner, the extravasation may be due to the fact that large arteries progressively narrow in to arterioles and then capillaries and tumour cells can be trapped in this small vessel, thus allowing the migration in the new organ (Nguyen 2004). Although the exact mechanism behind the tumour homing is not completely understood, recent results suggest that the selective homing of cancer cells may be due to three mechanisms: 1) presence in the target tissue of specific growth factors or appropriate extra-cellular matrix that favour the selective tumour growth, 2) presence in the target organ vessel endothelium of specific adhesive proteins that interact with the tumour cells, favouring the tumour invasion, 3) production of a chemotaxis soluble factor by the target tissue that attract the tumour cells ( Fidler 2003).
1.3.7 Genetic instability
Over the past 25 years numerous genetic alterations have been described in human and animal tumours. These genetic alterations can affect the DNA sequence and the chromosomes (Lengauer et al. 1998). The mutations of DNA include: substitution, deletion, translocation and insertion and they can affect one or more nucleotides. The necessity to transmit genetic information faithfully between generations demands genetic stability (Eisen and Hanawalt 1999)
In normal conditions the genome is affected by spontaneous mutations caused by physiological DNA instability and by imprecision of the DNA polymerase proofreading activity during the DNA replication (Alberts et al. 2002e). In eukaryotic cells, several enzymes have been described with DNA polymerization activity, and five are the most important DNA polymerases involved in DNA replication and repair, alpha, beta, gamma delta and epsilon. To date the only polymerase involved in mitochondrial DNA replication is polymerase gamma. In vitro studies on the fidelity of DNA duplication has shown that the nucleotide mis incorporation rate varies among polymerases, with one in 5000 bases for beta and one in 10 000 000 for delta and epsilon polymerases (Umar and Kunkel 1996; Loeb and Loab 2000). To avoid non-complementary nucleotide incorporation, polymerase delta, gamma and epsilon contain a proofreading activity (Kunkel and Alexander 1986). Normally DNA replication is carried out by delta polymerase, but recent reports show that in some tumours this priority is shifted in favour of less accurate polymerases, thus increasing the mutation rate (Loeb and Loeb 2000). Environmental agents such as ultraviolet light, ionizing radiations and toxic substances in the dietary uptake can induce mutations (Loeb and Loeb 2000).
1.3.7a Single Base Excision Repair
When a mutation effects on a single nucleotide then base excision repair take place. BER employs enzymes called DNA glycosylases, which are specific in removing a specific mutated base (Krokan et al 2000).
1.3.7b Nucleotide excision repair
The nucleotide excision repair (NER) system is able to repair DNA damage induced by UV. In contrast to BER, the NER system recognizes altered nucleotides by scanning the DNA for a conformational alteration (bulky lesion) (Wood 1996).
1.3.7c Mismatch repair
The mismatch repair (MMR) pathway includes a series of proteins that are involved in correcting errors that escape the DNA polymerase proofreading activity during DNA replication. They are also involved in suppressing recombination between non-identical sequences both in mitosis and meiosis (Kolodner and Marsischky 1999). Unlike BER and NER, MMR does not act on damaged or mutated sequences, but it targets only the newly synthesized DNA strand.
Inactivation of the MMR system produces microsatellite instability (MSI) (Atkin 2001).
1.3.7d. Homologous recombination
Homologous recombination repairs double strand breaks by using an intact and homologous DNA molecule as a template. In eukaryotes several proteins are involved in the homologous recombination process (Kanaar et al. 1998; Haber 2000).
1.3.7e. Non-Homologous End Joining
Non-Homologous End Joining (NHEJ) is the more important repairing mechanism when there is break in DNA double strand and it is very important mechanism in mammals (Khanna and Jackson 2001). During the NHEJ process small deletions are generated. Given that majority of the mammalians genome is composed of non-coding regions, the probability that in normal situations the NHEJ process induces mutation in genes is low (Alberts et al 2002e). However, if there are multiple break points NHEJ increases the occurrence of illegitimate recombination
(Rothkamm et al 2001).
1.3.7f Chromosome Instability (CIN)
The cell reproduces by a series of events that allow DNA replication and cell division in a process known as the cell cycle. In order to check the correct order of events that take place in the cell cycle, a complex cell-cycle control system has evolved (Alberts et al 2002c). This system checks normal cell cycle progression by a series of stage-specific sensors known as checkpoints that are able to induce the arrest of the uncompleted stage until it is completed.
The two fundamental processes in the cell cycle are the duplication and the division of the chromosomes, which take place during the Synthesis (S) and Mitosis (M) phase respectively. To prevent the possibility that two daughter cells have non-identical genomes, there are two checkpoints known as DNA replication and DNA damage checkpoints before mitosis, and one known as spindle-attachment checkpoint during mitosis (Alberts et al 2002c).
Chromosome instability (CIN) is also associated with structural alteration of chromosomes, which include reciprocal and non-reciprocal translocations, amplifications, deletions and insertions (Cairns 2005). Structural chromosome instability, resulting from DNA breaks and rearrangements, is due to alteration of cell cycle checkpoints, DNA damage response and telomere integrity (Gollin 2005). Structural alterations may results in altered gene expression or produce fusion or chimeric proteins with dysregulated or new properties (Greaves and Wiemels 2003). Studies have shown that a large proportion of human tumours with chromosome instability have a high rate of loss of heterozygosity (Rajagopalan and Lengauer 2004). Therefore it has been argued that chromosome instability could accelerate the rate of inactivation or activation of tumour suppressor genes or oncogenes respectively (Rajagopalan and engauer 2004).
CIN-associated genes can be classified on the basis of the mutations (Michor et al. 2005). Class I genes of CIN, e.g Mitotic Arrest Deficient gene (MAD-2 ) boost up CIN in case one allele is mutated or deleted. Class II genes of CIN e.g. Human Budding Uninhibited by Benzimidazoles (hBUB-1) gene boost up CIN if mutation is in one allele in a dominant negative fashion. Both Class I and Class II genes are required at the spindle assembly checkpoint (Amon 1999; Hoyt 2001). Class III genes of CIN e.g. Breast cancer gene BRCA1 and another Breast cancer gene BRCA2 boost up CIN if both alleles are mutated. BRCA genes have very important role at checkpoint and it is involved in DNA repairing and recombination (Yarden et al. 2002).
1.4 Evolutionary Dynamics of Tumour Development
According to clonal evolution theory, cancer is the result of somatic mutations selected during tumour evolution (Nowell 1976). It has been argued that tumour cells cannot acquire the mutations needed for tumour progression at a physiological mutation rate, but that the tumour cell must acquire an increased mutation rate (Cairns 1998; Loeb and Loeb 2000).
In order to induce cancer the mutations must affect a variety of genes that restrain somatic conflict (Frank and Nowak 2004). These genes are known as cancer related genes and can be subdivided in three categories: Gatekeeper, Caretaker, and Landscaper (Michor et al 2004). Gatekeeper mutations increase the cellular proliferation rate by the alteration of oncogenes, tumour suppressor genes and apoptotic genes (Michor et al 2004). Caretaker mutations increase genome instability by inactivating genes involved in maintaining genome integrity (Lengauer et al 1998). Landscaper mutations increase tumour proliferation by affecting genes involved in regulating the external cellular microenvironment (Bissel and Radisky 2001).
While mutations affecting oncogenes behave in a dominant way, because only one mutated allele can induce a tumour phenotype, mutations affecting tumour suppressor genes can be neutral if the normal allele compensates the mutant allele, disadvantageous if the mutant allele triggers apoptosis, and advantageous if the mutated allele is inactivated and the second allele is insufficient to balance the wild type allele (Michor et al. 2004). In small compartments the inactivation of the two alleles of a tumour suppressor gene, is unlikely, unless the mutation rate is increased by genetic instability (Nowak et al. 2005). Loss of heterozygosity increases with chromosome instability (Michor et al. 2004).
1.5 Tumours of Feline and Canine included in this study
1.5.1Mammary Tumours
Mammary gland tumours are most frequent in dogs (Moulton 1990) while in cats it is third in prevalence, after haemopoietic and skin tumours (Misdorp et al. 1999). The average age of peak prevalence of tumours in cats is approximately 9.3 years (Roccabianca et al. 2006). Mammary tumours can also affect male cats and dogs, with the average age for them being 12.8 years (Rutterman et al. 2000). Siamese has twice the risk in comparison to other breeds of cat (Weijer et al. 1972). Same predisposition was observed with our data, that all five cases collected in this study were belonging to Siamese breed. Mammary tumours are more prevalent in Pakistan and all the cats and dogs were between 5-11 years old. This suggests that there are more chances of mammary tumours in older cats and dogs. Mammary tumours included in this study were 23% all 22 tumours studied.
1.5.2 Canine Transmissible Venereal Tumours (CTVT)
Canine transmissible venereal tumours first reported by Blaine in 1810 (Blaine, 1810) is a transmissible cancer in dogs. Studies found that CTVT was transmitted by transplantation of living cells (Novinski 1876), confirming it as a transmissible cancer. CTVT is of clonal origin, originating from a founder dog 11,000 years ago (Katzir et al. 1985; Murgia et al. 2006; Rebbeck et al. 2009; Murchison et al. 2014). It is one of only two transmissible cancers known (Murchison 2008) and is spread by allogeneic transfer of cells between dogs, usually during coitus. It manifests as a tumour, associated with the external genitalia of both male and female dogs, although tumours can also arise in the mouth, nose or skin. It is purported to be of histiocytic origin (Mozos et al. 1996; Mukaratirwa and Gruys 2003), and usually remains rather localised, except for rare cases of metastatic spread.
Recorded cases of metastasis include involvement of the lymph nodes (Higgins 1966), skin (Dass 1986) and eye (Barron et al. 1963), among others. Experimental transplants of CTVT tumours into subcutaneous sites in experimental dogs are characterized by progressive and regressive phases. This is seen as a rapid volume increase, followed by tumour shrinkage, and eventually complete regression accompanied by serum-transferable immunity to reinfection (DeMonbreun 1934). In this project we collected 6 samples for BRCA1 and COX-2 studies in different tumours while 16 more samples for Department of Veterinary Medicine, University of Cambridge, UK. Although the prevalent rate of CTVT is in second number, many attentions were paid to collect CTVTs. For BRCA1 and COX-2 studies the 28% were CTVT out of 22 different tumours.
1.5.3 Perianal adenomas/Adenocarcinomas
There are many glands present around the anus of dogs. These are sebaceous and non-secretary glands, while anal sac glands are positioned at 4 and 8 o clock to the anus and secrete their secretions into the lumen of theanal track (Yang Hai-Jie et al. 2008). Perianal adenomas are more frequent than adenocarcinomas (malignant form). In this study 3 tumours were collected which were 14% of total canine tumours collected. These tumours are mostly common in medium to older age dogs.
1.5.4 Granuloma
Granuloma is also called as lick granuloma in dogs it is a type of skin cancer It typically results from the dog’s urge to lick the lower portion of one of her or his legs. This study reported 9% of total tumours included in this study.
1.5.5 Oral Tumours (Squamous cell Carcinoma)
Oral tumours are 4th common cancers in canines. Male dogs have 2.4 times greater risk of developing oral tumours than female dogs (Dorn et al. 1968). This study reported 9% oral tumours in a period of 2 years.
1.5.6 Lymphoma
Lymphoma is the second most prevalent intra –ocular tumours of dogs. Basic cause of lymphoma in dogs is unknown but genetic (chromosomal segregation), environmental and infectious factors such as retroviruses play vital role in developments of this cancer (Fighera et al. 2002). This study reported 9% Lymphomas of total collected tumours.
1.6 Rationale behind selection of genes
1.6.1 BRCA1 gene
BRCA1 gene is tumour suppressor gene, it is involved in repairing the DNA double strands breaks and in case of failure it leads the cells towards apoptosis (Starita. 2003). BRCA1 forms BRCA1 Genome Surveillance Complex (BASC) when it combines with different types of tumour suppressor genes, DNA damage sensors and signal transducers (Wang et al. 2000). It is involved in Ubiqutination, transcription regulation (Friedenson 2007; Friedenson 2008). In humans BRCA1 was first identified at chromosome 17 (Hall et al. 1990) and it was isolated in 1994 (Miki et al. 1994). It is present at 17q21 with a length of 100 Kb. In canine it is located on chromosome 9. BRCA1 has 22 exons in canines and felines; it encodes a protein of 1882 amino acids in canine and 1871 amino acids in feline. Many scientists from different research showed that women who have famililal mutations in the BRCA1 or BRCA2 (BRCA1/2) genes have increased risk of breast cancer (Struewing et al. 1997).
Fig 1: BRCA1 mechanism in DNA repairing. http://www.publichealthunited.org/leading-by-example-angelina-jolie-and-the-brca1-gene-mutation/
1.6.2 Cyclooxygenase-2 Enzyme (Prostaglandins, COX-2).
Cyclooxygenase-2 enzyme (Cox-2) is also called as Prostaglandins Endoperoxide synthase (PTGS). It is involved in the synthesis of prostaglandins which act as biological mediators in many body functions. It was first isolated from prostate gland that’s why it is called as Prostanglandin. Cyclooxygenase enzymes have two types, cyclooxygenas-1 and cyclooxygenase-2. Cyclooxygenase-1 is constitutively produced in the cell while cyclooxygenas-2 is inducible and it is constitutively produced only in kidneys, seminal vesicles and central nervous system. Its high expression has been recorded in many different types of tumours, it has been involved in anti-apoptosis, cell proliferation, tumour angiogenesis, cell invasion and immune suppression activities. In canine COX-2 is present on chromosome 7 having 604 amino acids and 10 axons. This correlation of cyclooxygenase-2 in cancer development suggests using new therapeutics against it. Studies have shown cycoloxygenase-2 high expression in number of different tumours (León-A 2008), such as intestinal, pancreatic, ovarian, prostatic, nasal cavity, oral cavity and mammary tumours of dogs (McEntee et al. 2002; Mohammed et al. 2004; Borzacchiello et al. 2007; Eplattenier et al. 2007; Mullins et al. 2004; Pireset al. 2010; Dore et al. 2003).
Fig 2:COX-2 mechanism of actionhttps://www.google.com.pk/search?q=cox+2+mechanism+of+action.
1.6.3 DLADQA1 (MHCII gene) (Additional work performed at Department of Veterinary Medicine, University of Cambridge, UK).
The Major Histocompatibility Complex (MHC) is a cell-surface protein mediating immune recognition through its interactions with T cells (Fig 3). There are three classes of MHC molecules in mammals - the classical MHC-I and II, and non-classical MHC-III Table 1). MHC-I interacts with CD8+ cytotoxic T cells, whilst MHC-II binds to CD4+ helper T cells. MHC molecules mediate antigen presentation to T cells. MHC-I typically presents self- peptides, whilst MHC-II presents foreign peptides. MHC molecules are extremely variable and polymorphic across the population, with a huge number of alleles at each MHC locus.
This allows MHC molecules themselves to behave as antigens in transplant rejection, with the graft MHC peptide recognized as non-self by the recipient, and thus rejected. It would be expected that CTVT, an allergenic graft, should be rejected for two reasons: host MHC will present tumour antigens as foreign non-self to the host immune system, and tumour MHC will present a mismatch to the host immune system as a foreign antigen itself (Fig 3). This project focuses on the DLADQA1 locus (Wagner et al. 2002), a classical MHC-II gene on dog chromosome 12. There is high level of MHC allelic variability in any population (Niskanen et al. 2013).
Fig 3:MHC is involved in graft rejection.
This rejection (represented by the red arrow) occurs according to two principles. Firstly, host T cells may recognize the host MHC presenting a foreign peptide that should activate an immune response. Secondly, host T cells would also be able to recognize the tumour MHC presenting any peptide as foreign, since it is not self-MHC. It is thus surprising that CTVT is able to persist as an allogeneic graft. MHC expression was previously characterised molecularly by Murgia et al through RT-PCR of a MHC-I (DLA88) and MHC-II (DLADRB1) gene (Murgia et al. 2006). They found that there was downregulation of expression of both these MHC genes. DLA88 showed low levels of tumour-specific expression, whilst there was no detectable tumour expression of DLADRB1. Murgia et al. also performed MHC genotyping for a number of CTVT samples and confirmed that all CTVTs shared the same haplotype (Murgia et al. 2006). They identified two clusters at the DLADQA1 locus, with some CTVTs appearing to be haploid the locus, whilst others remained diploid. This is in contrast to evidence that suggests the DLADQA1 locus had undergone a copy-neutral loss of heterozygosity (LOH) (Murchison et al. 2014).
1.6.4 Technologies used in this research work.
Different technologies are being used in cancer research such as PCR, flow cytometry, immunohistochemistry (IHC), in- situ hybridization (FISH, CSH) and microarray for diagnosis (Pawanet al. 2010). Here, I used Real time PCR for gene expressional analysis of BRCA1 and COX-2 and DLADQA1 (MHCII). Histopathology (Hematoxyline and Eosin staining) was performed for the diagnosis of tumours. CTVT diagnostics qPCR was also performed to measure the allele’s quantity of LINE-myc gene and CDKN2A gene. Conventional PCR measures at End-Point, while Real-Time PCR collects data during the PCR shows the data and quality of data during exponential growth phase also it has increase dynamic range of detection, it is very sensitive and no need for post PCR processing. Immunohistochemistry was performed to find out the expression of MHCII antigens in CTVTs. The serum protein electrophoresis and serum biochemistry was also measured. Western blotting was performed to detect antibodies in CTVTs (protein expression). It is a very good technique to measure the gene expression at protein level in fluidic material of cells. We performed capillary electrophoresis to find the mutations/SNPs in our genes of interest (BRCA1, COX-2 and DLADQA1). Genetic analyzer was used to find the sequence variations in our genes of interests. Other methods used for sequence variation studies, like SSCP, DGCG and HPLC miss the mutations (Rassi 2009). So the sequencing by capillary electrophoresis was the best option for this study.
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50.
Development Of DNA Based Diagnosis Of Ancylostoma Caninumin Dogs And Its Specificity With Traditional Fecal Microscopy
by Abida Rehman (2012-VA-648) | Dr. WasimShehzad | Mr. Akhtar Ali | Dr. Imran Rashid.
Material type: ; Literary form:
not fiction
Publisher: 2014Dissertation note: The blood feeding, canine hookworms have gained importance due to their potential to cause a variety of diseases in animals and human communities (Traversa 2012). Almost all types of canine hookworms are involved in causing zoonotic diseases(Traub et al. 2008), infecting more than half a billion people worldwide (Fenwick 2012), which result in ~65000 deaths annually (Plotkin et al. 2008). Ancylostoma caninum is the most prevalent and pathogenic intracellular obligate hookworm parasite of dogs (Bojar and Klapec 2012). In Pakistan, parasitic infection byA. caninum is widely prevalent with variable distributions in different parts of the country. A microscopic based study in the Lahore areashowed 59.1% ofA.caninuminfestation in dogs (Ashraf et al. 2008).
Clinically,A. caninum has been responsible for often neglected disease ancylostomiasis in its host.In this condition, it principally attacks on the mucosal layer of small intestine through its buccal capsule for sucking blood (Marquardt et al. 2000). Secretory anticoagulant proteins of A. caninum help in this process by blocking a wide variety of blood clotting factors including Xa. The inhibition of blood clotting factors causes greater blood loss which ranges from 1-2ml/worm/day (Cappello et al. 1995; Georgi et al. 1969; Stassens et al. 1996). The disease is indicated by the symptomsof weight loss, lethargy, roughness of the hair coat,infected pale mucous membranes, tarry feces and excretion of eggs (Marquardt et al. 2000).In chronic situation,A. caninum producesa devastating condition of iron deficiency anemia with intestinal bleeding (Loukas and Prociv 2001).A. caninum infection in the dog isaccompanied by other life threatening pathological conditions, these includegastrointestinal infections, hypoproteinemia, mental retardation,pneumonitis andacute fatalities (Schwenkenbecher and Kaplan 2007).Especially, puppies are more suscepted to aforementioned diseases because of transmammary transmission,low levels of body immunity and higher egg count(Anderson 2000; Olsen 1986).
The life cycle of A. caninumis almost same both in human and dog. Itis most complex and critical for them as compared to other members of its genus. The cycle starts with the production of eggs by adult worms within intestine of an infectedhost which are passed out with feces. These eggs survive in soil without damages by variable environmental conditions,can become a source of reinfection for host species. Theinfective filariform larvae (hatch from eggs) get their route ina host bodyby penetrating through hair follicles on skin contact. In puppies these are mostly transmitted through transmammary or prenatal routes. After penetration, these larvae take way to lungs through the blood or lymphatic circulation. From here, these can be swallowed towards intestine where they attached to feed on blood and mucous (Marquardt et al. 2000). They also migrate to skeletal muscles via somatic circulation where they depositedas hypobiotic larvae. Warm and moist conditions cause their reactivation and migration towards gut (Prociv and Luke 1995; Traub et al. 2014).
It is seenthat a healthy pet doggetsA. caninuminfection mainly due to lackof proper veterinary care, large population of infected stray dogs, poor sanitation, and by contamination of public parks and streets (Klimpel et al. 2010; Zewduet al. 2010). Infective dogs are responsible for transmission of this parasite to humans either playing role aspets, stray or rescue animals (Jafri and Rabbani 1999; Szabova et al. 2007) by shedding millions of eggs(Epe 2009). Children areparticularly and frequently attacked byA. caninumbecause they used to play in open contaminated areas (Farooqi et al. 2014).
Transmission of this parasite to humans is mostly by penetration through the skin, when it comes in contact with its filariform larval stages present in contaminated soil.Humans also get infection through larval ingestion, and larvae canbe transmitted from the fur coat ofinfected companion animals (Caumes 2000; Hochedez and Caumes 2007; Provic 1998). In human, penetration of A. caninumfilariform larvae causessevere cutaneous larva migrans (CLM). It is the most devastating hypersensitivity reaction, characterized by long follicular, pustular, ephemeral lesions at infection sites with intense itching and pain (Kirby-Smith et al. 1926; Little et al. 1983). These sites can be attacked by bacterial species leading tothe damages ofsoft tissues (Chaudhry and Longworth 1989). CLM caused by A. caninum has achievedmore attentionmainly due to associated pathological conditions. These include eosinophillic enteritis, pneumonitis, and growth defects, etc. (Bowmanet al. 2003; Garcia et al. 2008; Tu et al. 2008). In childrenheart problems and mental retardation also havebeen reported (Albonico et al. 1999; Crompton 2000).
As, A.caninumiscausing serious healthhazards, so improved and proper control of its pathogenesis is mandatory.The accurate diagnosisof infection helps to prevent its transmission by selection of appropriate precautions and vaccines. Specific identificationis alsohelpful to minimizedthe chances of anthelminticdrugresistance by A.caninumas reported in different studies (Bethony et al. 2006; Kopp et al. 2007; Peeling et al. 2008; Roeber et al. 2013).
Different diagnostic methods are used to identify parasitic hookworms. Currently, fecal microscopy is the widely applied diagnosticmethod for the identification of hookworm infection,which depends on morphological based analysis of eggs (Katz et al. 1972; Ngui et al. 2012b). The main advantage of this traditional approach is its tendency to analyze sampleboth qualitatively (floatation, sedimentation) and quantitatively (McMaster, FLOTAC, Katokatz)(Cringoli et al. 2011; Eberl et al. 2002). However, the identifications based on morphological characteristics may prone to inaccurate diagnosis, because eggs of many hookworms e.g. Ancylostoma, Trichostrongylus, Unicinariastenocephala, Oesophagostomum, Necator americanus and Terniden species are morphologically indistinguishable,thiscan variate specificity and sensitivity of microscopy (Bajwa et al. 2014; Monis et al. 2002;Tan et al. 2014).
Improvement in microscopic detection can be made by usingcopro-culture and immunodiagnostic techniques especially when results are confounding. In the copro-culture method, eggs are raisedto larval stages in appropriate growth conditions in the laboratory andthen classified up to genus level (Reiss et al. 2007). But in this technique as microscopic examination, morphological similarities between larval stages of related species hampers specific identification. Moreover, it is time consuming (takes about 7-14 days) and requires experienced technicians to handle larvae. Similarly, it is very difficult to evaluate exact burden of worms as there is significant variations in the number of excreted eggs in chronic cases, which is another reasonable disadvantage ofthis diagnostic technique (Booth et al. 2003; Schar et al. 2013). Immunological assays, including ELISA,based on the detection of coproantigens in the feces or serum of the infected dog using captured IgG have been used to increase the sensitivity of analysis (Kwon et al. 2003;Loukas et al. 1992). These methodsallowed effective and quick detection of parasistes as compared to fecal microscopy and coproculture technique.But issue of cross reactivity with other antigens like of Strongyloides stercoralis in mixed infections isan associated problem (Lindo et al. 1994). Moreover, immunological assays failed to provide information about past or current infection and also do not differentiate between species in mixed infection (Basuni et al. 2011).
All the problems of traditional methods can effectively addressed by the adoption of DNA based molecular approaches. These methods have beenproved as worthwhile alternatives to identify parasitic species in any developmental stage (Muldrew 2009; Ndao 2009; Vasoo and Pritt 2013). Specifically, these techniques are very feasible for specific identification of genusAncylostomabecause ithasambiguous features.Advanced highly sensitive DNA based diagnostic procedures have potential to identify the causative agentfromminute quantity of DNA (from 0.2g of egg), as in low worm burden with no symptom of disease(de Carvalho et al. 2012; Wong et al. 2014).Rapid detection of parasites can be made in only one day even in case of mixed infections(Gasser et al. 2009).
There are various available approaches in DNA based methods which can be applied for the detection of Ancylostomaspecies, theseinclude PCR-RFLP (e Silva et al. 2006; Traub et al. 2004), copro-PCR (Sato et al. 2010), single-strand conformation polymorphism (SSCP) (Gasser and Monti 1997; Monti et al. 1998),specific (conventional) PCR (Yong et al. 2007) and multiplex real time PCR (Jonker et al. 2012). The conventional PCR techniquehas worldwideapplications in specie specific identification of parasiticspecies(Gordon et al. 2011). For specific diagnosis,genetic markers have been identified in mitochondrial (cox1 gene) and nuclear genomes (internal transcribed spacers (ITS-1, ITS-2), 5.8S and 28S in ribosomal DNA (rDNA)) (Chilton 2004; Denver et al. 2000; Gobert et al. 2005; Ngui et al 2012a). Preferably, spacers are more suitable for diagnostic purposes because these regions havesequence variationsamong different species. Moreover, these are shorter in length (250-300) in comparison with mitochondrial DNA (Blouin 2002; van Samson-Himmelstjerna et al. 2002). Therefore, the availability of sequencing technologies,specific genetic markers and large amount of genetic data have increased the chances of implementation, development and effectiveness of DNA based diagnostic method for identification ofA. caninum(Taniuchi et al. 2011).
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