Phylogenetic Analysis Of Major Fresh Water Carps Of Pakistan Through DNA Barcoding
Material type: Book ; Literary form:
Publisher: 2014 Dissertation 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.
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
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).
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
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).
Development Of DNA Based Diagnosis Of Ancylostoma Caninumin Dogs And Its Specificity With Traditional Fecal Microscopy
Material type: Book ; Literary form:
Publisher: 2014 Dissertation 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).
Availability: Items available for loan: UVAS Library [ Call number: 2262-T] (1).
Application Of Microsatellite Markers For Genetic Diversity Analysis Of Endangered Punjab Urial (Ovis Orientalis Punjabiensis) In Pakistan
Material type: Book ; Literary form:
Publisher: 2014 Dissertation note: Biological diversity is now recognized as common concern of mankind and genetic diversity is the major driver of variation within and across breeds, which helps populations to adapt to environmental changes. There is very little importance is given to conserve wild sheep in previous years and its genetic diversity is decreasing day by day. Every now and then breeds are being haunted for crossing to some other imported breed without attempting to see if such efforts will be sustainable. For any breed development efforts thus, available genetic resources need to be characterized both at phenotypic and genetic levels (Khan et al. 2007).
Among the three levels of Biodiversity, one is Genetic variation which is suggested bythe International Union forNatureconservation (IUCN) for preservation (Mc Neely et al. 1990). The reason for it is that firstly; genetic diversity favors the changes as the environment changes and secondly; it prevents inbreeding depression (Reed and Frankham 2003). In this way genetic diversity increase the survival status and increase fitness of individuals.
Among many other wild animals present in Pakistan, there are 6 to 9 species of wild sheep (Ovis orientalis) are present which have different color and size of their winter neck ruff of males, saddle patches and horns color. Urial is a picture of Marco Polo in texture and hue. In Pakistan, ladakh urial, Blanford urialand Punjab urial are found in Gilget, Baluchistan and Punjab respectively. The discrepancy lies in the color of ruff among these 3 sub species (Roberts, 1977). Urial is among those precious wild animals that were hunted severely for trophy and other purposes in the past, that’s why included in red list of IUCN in vulnerable category (IUCN 2000).
Punjab Urial (A type of wild sheep – Ovis vignei punjabiensis) belongs to family bovidae which is the large family consisting of 140 species (Glazko et al. 2011) is facing serious threat of extinction in Pakistan is a medium-sized wild sheep which is included in IUCN red list of endangered animals (IUCN 2002). Urial is inhabitant in Western Central Asianregion stretching from northeast side of Iran and west side of Kazakhstan to Balochistan (Pakistan) and Ladakh regions of North India. The local name of Urial is Shapo, Arkar and Gad. Reddish-brown outstretched pelt that achromatizes during the winter is one of the distinct traits of urial (Aleem 1977; Schaller 1977). Urial is gregarious and sexually dimorphic as males are called rams and females as eves (Awan 2001). Male have weightof 40 kgand have large spiralled horns having height of 80 to 100 cm and females have comparatively less weight and height of 25 kg and 12 cm respectively and have uncurled horns.Females give birth to 1 or 2lambs in recent days of April (Awan, 2001). Males have a black ruff expanded from the neck to the trunk and notably longer horns. Table 1.1:Some physical features of the Punjab Urial (Awan et al. 2001)
Body weight 40 kg male; 25 kg female
Shoulder size 31-35 inches or 80-90 cm
Horn size 39 inches or 80-100 cm long male; 12 cm long female
Urial are found in moderate to very arid habitats, especially grasslands including agricultural fields and woodland areas (Valdez, 1982). Urial is herbivorous and eats grasses, shrubs and grains. The patch of salt range of Pakistan which fall in the area of Pind Dadan Khan, Choa Saidan Shah and Kallar Kahar is considered like a paradise on earth for the wild fauna. The fascinating hills of these areas are covered with thick trees and different wild plants are also the sanctuary of urial.
Table 1.2: The details of these sub species in IUCN list of endangered mammals (IUCN 2002).
Subspecies Citation in IUCN list
Ovis vegnei blanfordi VUC 1 Appendix ll
Ovis vegnei punjabienses ENA1cde,c1+2a Appendix ll
Ovis vegnei vegnei VUC 1 Appendix ll
In Pakistan, Punjab Urial dispersed throughout the Kala Chitta and Salt Range in a very little number(Hess et al, 1997). The Afghan Urial inhabits Baluchistan, Khyber Pakhtunkhwa, and Sindh Provinces. In Chitral District, little segregated populations of Ladakh urial are stillextensively distributed near the west bank of the Kunar river from Chitral southwards to Drosh. Ladakh Urial existence at the east bank of Kunar river and north of Chitral are not proved (Malik 1987). Total population estimate of urial is recorded by conducting several surveys. Reasonably 2,500 to 3,000 urial existsin Baluchistan (Hess et al. 1997). In 1993 the overall population assessment of Northern Areas was four hundred to five hundred Urial (Rasool 1999). In Pakistan, there were seemingly less than 600 Ladakh urial (Hess et al. 1997;NWFP 1992; Schaller 1971, 1977) but the number of urial decreased to 200 to 300 urial in all over northern areas (Rasool 1999). Based on the facts mentioned above there is dire need to conserve the Urial population in the country. The urial is one the precious fauna of Pakistan and provides us with wool and meat (fat, flesh or any eatable part). It is also important in economic way and in maintaining of ecosystem balance. In the beginning, sheep were reared for meat, milk and skin (Ensminger and parker, 1986). After 3500 B.C. men learnt to spin wool and so used wool in textile industry (Smith et al. 1997). So, because of increasing world population, there is great demand of these products and increasing day by day. That’s why we have to conserve is natural resource of Pakistan.Habitat fragmentationleads to the risk of exaggerated genetic drift and inbreeding in isolated population. So, there is need to save urial from these threats by enforcement of law and conserving it for future.
In all over the euchromatic genome Microsatellite markers are present. These markers are highly polymorphic (Ellegren 2000; Schlotterer 2000).A lots of polymorphic microsatellites have been analyzed in ruminants like domesticsheep, cattle etc. (de Gortari et al. 1997,1998 ;Hayes et al.1996; Jenkings et al. 1997) aiding the use of these in parentage testing.
Microsatellite markers are among the most reliable molecular markers for genetic characterization studies in animal species (Sunnucks, 2001) and are simple sequence repeats (SSRs) of 1-6 base pairs, repeated tandemly in coding as well as noncoding portion of DNA in prokaryotes and eukaryotes (Weber and May, 1989; Toth et al., 2000). Microsatellite markers have often used for genetic diversity studies because they areabundant, unbiased, widely distribution all over the DNA, highly polymorphic, easy in assessment like genotyping of these markers (Canon etal. 2001).Microsatellite markers aid in genetic differentiation and conservation studies (Peter et al. 2007;Rendo et al. 2004; Arranz et al. 2001).These are considered very useful markers for assessment of genetic diversity, parentage confirmation, genome mapping, disease research population genetic studies and conservation genetics. These are also reported to be efficient enough to identify within and among breed differentiation and population sub structuring in cattle (Glowatzki-Mullis et al. 1995; Ciampolini et al. 1995; Garcia-Moreno et al. 1996; Jarne and Lagoda, 1996; MacHugh et al. 1998).Therefore the conservation activities are very important to save Punjab Urial from extinction and the study is designed to explore its genetic diversity.
Availability: Items available for loan: UVAS Library [ Call number: 2261-T] (1).
Genetic Characterization Of Pakistani Wild Quails Using Mitochondrial Coi Gene
Material type: Book ; Literary form:
Publisher: 2015 Dissertation note: A latest taxonomic tool termed as DNA barcoding is being used to genetically characterize animals. DNA barcoding is helpful in determining evolutionary relationship among species. Being a small sized genome and agile enough to show rapid mutation, mtDNA has been used as a pertinent marker of molecular biodiversity. DNA barcode works as an efficient tool in food manufacturing industry, diet investigation, forensics analysis, preventing unlawful trade and felonious poaching. The aim of this study was to develop DNA barcode for genetic characterization of Pakistani wild quail. Pakistani wild quail is important due to its demand for eggs, meat production, experimental purposes and gaming as well. Japanese quail was also included in this study because this quail is excessively produced in Pakistani farms. Japanese quail is present throughout the year and is comparatively bigger in size than wild quail. It has longer lifespan; farmers can easily breed this species in farms. It is suitable in poultry due to better meat yield.
COI gene (500bp) was used as a molecular marker for identification at species level. DNA was extracted from blood samples of ten wild quails (Coturnix coturnix and fifteen japanese quails (Coturnix japonica). Reported bird universal primers were used to amplify COI region from the extracted mtDNA samples using PCR. Amplicon were then sequenced by Sanger sequencing method (Sanger et al. 1977). Forward and reverse DNA Sequences were aligned with the reference sequence using nucleotide BLAST on NCBI to observe the dissimilarity among the sequences. Consensus sequences generated were used to construct their phylogenetic tree to see their evolutionary relationship with other bird species. Japanese quail which is thought to be domesticated from Japan, its Pakistani population showed close relation with sequences
generated in Japan for this particular species. Pakistani wild quail species showed its closest linkage with C. coturnix.
In conclusion, COI barcode proved as an authentic tool for species identification and phylogenetic inference of Pakistani wild and farm grown quails. Wild quail species has been characterized using partial COI gene sequences. This study has provided a specific genetic marker which can differentiate Japanese quail from wild quail at molecular level as most of the time both species are confused with each other. It can be helpful to the farmers and bird fanciers because they can select the birds of their choice correctly. This is the first study reporting DNA barcode of this Pakistani quail species. It would help researchers to study about phylogenetic and taxonomic status and also assist quail fanciers and quail farmers to unaffectedly identify their species of interest in farming. Identification of quail species is also important for conservation of biodiversity as it helps in preservation and identification of endangered species by generating their barcodes from even minimal evidence available.
Availability: Items available for loan: UVAS Library [ Call number: 2311-T] (1).
Antiviral Effect Of Human Saliva Against Avian Influenza Virus Strain H9n2
Material type: Book ; Literary form:
Publisher: 2015 Dissertation note: Saliva is an important body fluid that contains a complex array of proteins, peptides and various substances that help in maintaining the health of the oral cavity. Saliva exhibits a broad-spectrum of antiviral activity against enveloped viruses as it disrupts the viral membrane. Influenza is a common virus that has been diagnosed in humans and avian species due to AIV. This study has demonstrated the naturally occurring antiviral activity of human saliva against the H9N2 influenza virus that serves as a serious threat to poultry and has been shown to possess high zoonotic potential which can cause a new pandemic.
In this study saliva samples from healthy individuals were taken and the natural antiviral ability of saliva was observed against AIV (Pk-UDL/01/08 H9N2) of calculated EID50 106.66. Inoculum prepared from saliva and H9N2 virus was injected in 9 days old embryonated eggs using CAS route and incubated at 37°C for 48 hours. A negative control (only saliva) and positive control (only virus inoculum) was also determined in the current study. The antiviral activity of saliva was observed through haemagglutination test. The HA test of harvested fluid showed that human saliva indeed possesses antiviral activity against H9N2 virus and can be used as a natural antiviral agent in medicine.
Furthermore, the genomic DNA was extracted from the blood samples. HTN3 gene responsible for histatin production, was amplified using gene specific oligonucleotides. The obtained HTN3 gene sequences were analyzed using Chromas software. The sequence alignment showed 99% similarity to the available sequences in NCBI database and 100% similarity to each individual sample. To conclude, this study has demonstrated that human saliva possesses antiviral activity against H9N2 virus. The nucleotide sequence analysis from each sample
showed no particular change which shows that antiviral activity of glycoproteins present in saliva does not vary at a genetic level. This innate antiviral activity can open a new frontier when it comes to combating viral infections that have grown resistant to conventional drugs in both human and animal subjects.
Availability: Items available for loan: UVAS Library [ Call number: 2336-T] (1).
Mutational Screening Of The RB1 Gene In Pakistani Patients With Retinoblastoma
Material type: Book ; Literary form:
Publisher: 2015 Dissertation note: Retinoblastoma is a neonatal intraocular tumor caused by biallelic inactivation of RB1 gene. Rb
patients and asymptomatic carriers undergo a series of clinical tests for diagnosis and tumor
treatment. These clinical examinations prove to be expensive and time consuming. On the other
hand if the proband’s RB1 gene mutation status is determined by genetic testing, it can prove as
more significant and cost effective diagnostic methods. Secondly, only those asymptomatic or at
risk carriers with the mutation, require clinical surveillance while those proven to be unaffected
do not require additional clinical examinations. Furthermore early diagnosis of Rb by molecular
testing can enable and enhance clinical management, earlier treatment, follow-up care, carrier
screening, genetic counseling, prenatal diagnosis and reproductive planning in predisposed
families. Irrespective of the importance of molecular testing of Rb patients, in Pakistan only a
few clinical reports on Rb are available so, there was a dire need to find RB1 mutations in
Pakistani Rb patients and to set a molecular based diagnosis for poor affected families. Keeping
in view the importance of molecular diagnosis, in this study a reliable genetic test has been
developed to detect the RB1 germline mutations in Pakistani Rb patients.
During this study, 70 Rb patients including 38 unilateral and 32 bilateral cases were enrolled,
from different regions of Pakistan. By using direct sequencing method, seven novel and twelve
reported RBI mutations were found. The novel mutations included three frameshift mutations
(c.1116_1119delCACT in exon 11, c.1436_1437delAC in exon 16 and c.2060_2061insTCATT
in exon 20) and four substitutions (c.148G>T in exon 2, c.610G>T in exon 2, g.94G>C in exon
7, c.947A>T in exon 10 and g.1991G>C in promoter region) while twelve reported mutations in
22 patients included, 9 substitutions (c.160G>T in exon 2, c.289G>T in exon 3, c.751C>T in
exon 8, c.920C>T in exon 9, c.967G>T in exon 10, c.1072C>T in exon 11, c.1654C>T in exon
17, c.2063T>C in exon 20 and c.2359C>T in exon 23), one frameshift mutation (c.772_776del in
exon 8) and two splice site mutations (c.380+1G>T and c.1215+1G>A in intron 3 and 12
respectively). Mutation detection rate was found to be 77.8% in (7/9) bilateral familial, 50% in
(2/4) unilateral familial, 56.5% in (13/23) bilateral sporadic and 14.7% in (5/34) unilateral
sporadic patients while overall rate of mutations in bilateral and unilateral patients was detected
as 62.5% (20/32) and 18.4% (7/38) respectively. Beside mutations one novel c.940-64C>T
(intron 9) and nine reported intronic variants c.380+45 C>T (intron 3), c.501-77G>A (intron 4),
c.1128-72T>G (intron 11), c.1695+99A>T (intron 17), c.1695-1696delAA (intron 17), c.1815-
104A>G (intron 18), c.1961-10T>C (intron 19), c.2663+33T>C (intron 25) and c.2664-10T>A
(intron 25) were also found. Carrier screening facility was also provided to six asymptomatic
siblings (as possible carriers) of familial proband but none of them was found to be diseased.
Hopefully, in future the findings and developed protocol of this study will help to reveal the
molecular basis of Rb in Pakistani Rb patients which additionally help to secure vision and life
of Rb patients. Further, in Pakistan there is dire need to develop “National Rb Registry Centre”,
to register all new Rb cases for finding incidence rate and prevalence of Rb in Pakistan. Beside
this other related issues like financial constraints, health education, planning and awareness
about Rb, occupational training for health providers, capacity building for neonatal
ophthalmologic screening and cosmetic rehabilitation for surviving Rb patients are important and
Availability: Items available for loan: UVAS Library [ Call number: 2370-T] (1).
Molecular Characterization Of Local Donkey Based On Mitochodrial D-Loop Analysis
Material type: Book ; Literary form:
Publisher: 2014 Dissertation note: Research on control region or mitochondrial d-loop is of special interest in all mammalian species because they the displacement loop hyper-variable region of mtDNA (D-loop), which is of 1200 bp, is very important for genetic variations. It is also important for amtDNA in Nuclear Mitochondrial sequences (NUMTs) in vertebrates species so that it rarely occurring NUMTs. The control region in equine consist of two highly variable regions (HVR1 and HVR2), 4 conserved blocks (CSB), and variable repeats of 8 bp motifs (Cothran, et al. 2013).
The displacement loop hyper-variable region of mtDNA (D-loop) of Pak- local donkeys are very similar to genome of other species; however there are considerable differences in the mtDNAevolutionary rate for different taxonomic groups.
The phylogenetic tree based on consensus sequences of 12 Asian Donkeys breeds available on NCBI and sequences of local Pakistani donkey breeds showed their genetic relationship among each other. The clade was consisting of Pakistani local donkey breeds i.e. 3-SF, 7-SF,10-SF,11-SF,12-SF,13-SF, K-3,K-6,K-11,K-13,K-16,K18,K-19 and K-20 showing their high relatedness.K-8 and 8-SF have more mutation rate in the sequence and have more diversity from other individuals.
The second major branch was furher divided in two sub branches i.e. Donkey family representing Equusburchellichapmani (JX312729), Equusburchelliquagga (JX312733), Equusgreyvi(NC020432) and Equus zebra (JX312718) clustered together. The second sub branch was consisting of other donkey breeds i.e. Equushemionusonager (JX312730), Equushemionus(NC016061) and Equushemionuskulan (NC018782) grouped together while two Pakistani local donkey breeds i.e. SF-8 and K-8 were clusterd with Equusasinussomalicus (AP012271) and Equusasinus (X97337).
The d-loop sequence of Human (Homo sapiens) was taken as out group and it clearly differed and separated from rest of phylogenetic tree i.e. camel, bovine, ovine, caprine and other mammals.
The phylogenetic tree constructed under this study not only confirmed the status of Pak- local donkey breeds but also confirmed the genetic relationship among other mammalian species, thus reconfirming the already established biological classification.
Availability: Items available for loan: UVAS Library [ Call number: 2362-T] (1).
Genetic Identification And Molecular Classification Of Sub-Family Phasianinae Of Pakistani Bird Species Through Dna Barcoding
Material type: Book ; Literary form:
Publisher: 2015 Dissertation note: DNA barcoding is a precise technique that uses molecular genetics tools for accurate identification, categorizing, relating and separating the phylogenies of species. Being a small sized genome and agile enough to show rapid mutation, mtDNA has been used as a pertinent marker of molecular biodiversity.The aim of this study was to develop DNA barcode for genetic characterization and classification of Sub-family Phasianinaeof Pakistani bird species. Theyhave not been genetically identified yet in Pakistan. It includes birds like domestic chicken(Gallus gallusdomesticus), aseel chicken(Gallus gallusdomesticus strain),blue peafowl(Pavo cristatus), green peafowl (Pavo muticus), white peafowl (Pavo cristatus leuticus), Kalij pheasant (Lophura leucomelanos),monal pheasant (Lophophorus impejanus),koklass pheasant(Purcrasia macrolopha), ring necked pheasant (Phasianus colchicus), Tragopan (Tragopan melanocepals) andred junglefowl (Gallus gallus). These birds are considered an important part of an ecosystembecause they play a significant role in seed dissemination, pollination of plants and disease spread which are the basic constituents of an ecosystem. They are used for food, hunting and entertainment purposes.
Mitochondrial geneCytochrome c oxidase subunit 1 (CO1)of 500bps was used as a marker for identification at specie level.Genomic DNA was extracted by each blood and tissue sample of eleven bird species (33 samples). Amplification of CO1 gene was a done by using a universal set of primers (BIRDF1 and BIRDR1)containing region of almost 750 bps (Hebert et al. 2003).Amplicons were purified and sequenced Sanger sequencing method (Sanger et al. 1977). Forward and reverse sequences were analyzed using softwaresEMBOSS merger,ClustalW, BioEdit and nBLAST. Phylogenetic analysis of selected bird species was done. Each sequence was aligned
with its reference sequences of CO1 gene present on NCBI. Every nucleotide position which did not align with the reference sequence was studied to identify SNPs. Fixation index (FST) were used to measure species diversity within a same sub population relative to that found in the entire population. Consensus sequences (500bps) generated was used to construct their phylogenetic tree to see their evolutionary relationship with other bird species. All species showed their closest linkage with their respective species. Pakistani population of peafowl and chicken species showed the close relation with same sequences generated in China. Tranopans showed its closest linkage with T. temminckii.
In conclusion, seven species ofPhasianinaesub-family of Pakistani bird species was genetically characterized first time in Pakistan by using CO1 as a barcode. It proves that DNA barcoding is an efficient and accurate molecular tool for species identifica¬tion and phylogenetic implication. This study leads to establish a DNA Data Bank that helped scientists to investigate the biodiversity, taxonomic classification, specie identification, in forensic purposes and to study the genetic and phenotypic evolution of these species. DNA barcoding through CO1 gene works as a functional tool for detectingmeat mislabeling and preventing illegitimate trade. This study has established foundations for molecular biologists to study taxonomic uncertainties at sub species level using SNP based identifying marker. It helps in preservation and identification of endangered species by generating their barcodes from even minimal evidence available.
Availability: Items available for loan: UVAS Library [ Call number: 2376-T] (1).
Molecular Characterization of Pakistani Common Leopard
Material type: Book ; Literary form:
Publisher: 2015 Dissertation note: CD not available.
Availability: Items available for loan: UVAS Library [ Call number: 2379-T] (1).
Expression And Purification Of A Potent Surface Antigen (Sag1) Of Toxoplasma Gondii In Prokaryotic Expression System
Material type: Book ; Literary form:
Publisher: 2015 Dissertation note: Toxoplasma gondii, an intracellular obligate parasite infects almost all warm-blooded animals including human. Toxoplasmosis, caused by T. gondii, may show minute to severe clinical results in humans. Currently, there is no vaccine available for human use. SAG1 is a major candidate of interest for vaccine development that elicits humoral as well as cellular immune response against this devastating parasite.
rSAG1 that had already been ligated in pET28/His expression vector, was transformed in E. coli (BL21) host and expression was confirmed by means of SDS-PAGE and western blotting. Nickel columns were utilized for affinity based chromatographic purification of rSAG1. This purified protein was then quantified via protein quantification kit. Immunogenic recombinant SAG1 can be used in diagnostic antigen-antibody tests e.g. in ELISA. Moreover, it might be used in vaccination against T. gondii. Vaccine against this parasite may alleviate socio-economic burden on human society that ultimately modulates the health parameters for better living.
Availability: Items available for loan: UVAS Library [ Call number: 2393-T] (1).
Genetic Identification And Characterization Of Pakistani Birds Of Perdicinae Subfamily (Partridge) Through Dna Barcoding Method
Material type: Book ; Literary form:
Publisher: 2016 Dissertation note: Pakistani birds of Perdicinae sub family are cage and game birds. Birds includes Altectoris chukar, Ammoperdix heyi, Ammoperdix griseogularis, Francolinus francolinus and Francolins pondicerianus. Traditional methods of identification were based on the phenotypical characterization of birds, which may lead to incorrect identification, so there was need to explore their characters at DNA level for accurate identification and to establish a DNA reference.
Birds of sub-family Perdicinae have not been genetically characterized in Pakistan. A new precise method “DNA barcoding” was applied using COI gene of mDNA for authentic identification and classification of these birds. Blood and tissue samples of five species (fifteen samples) were obtained. DNA of each sample was extracted by organic method. Amplification of CO1 gene was done by using a universal set of primers BIRDF1, BIRDR1. Sequence of 450bp were analyzed using bioinformatics softwares. Each sample was aligned with its reference sequence of COI gene available on NCBI. Every nucleotide position which did not align with the reference sequence was studied to identify SNPs. A common phylogenetic tree of all partridges showed that they have common ancestor about 0.7 million year ago, F.francolinus, F.pondicerianus and A.heyi shared a common clade whereas A.chukar made a separate clade from the ancestor. A.heyi and F.pondicerianus showed closed resemblance. It has been proved that DNA barcoding is an efficient and accurate molecular tool for species identification and phylogenetic implication. This study established a DNA Data Bank that helped scientists to investigate the biodiversity, taxonomic classification, species identification and also established foundations for molecular biologists to study taxonomic uncertainties at sub species level using SNP based identifying marker.
Availability: Items available for loan: UVAS Library [ Call number: 2714-T] (1).
Association Of Tryptophan Hydroxylase 2 Gene Polymorphisms With Risk Of Depression In Homo Sapiens And Equus Caballus
Material type: Book ; Literary form:
Publisher: 2017 Dissertation note: In the biosynthetic pathway for brain serotonin, Tryptophan hydroxylase-2 (TPH2) acts as the rate-limiting enzyme. It is a key element in maintaining adequate serotonin neurotransmission in the central neuron system (CNS). It is broadly discussed as an important candidate gene in multiple psychiatric disorders, especially suicidal behavior and depression. A relationship between TPH2 and major depressive disorder (MDD) has been reported by multiple gene-disease association studies in different populations. Horse can be employed as a valuable candidate for an animal model of depression because it shares environmental factors which are known to cause depression in humans.The hypothesis of this study was that, there is association between TPH2 gene polymorphisms and risk of developing MDD.Blood was collected from each participant.Human (Experimental and Control group) and Horse (Experimental and Control group).DNA wasextracted usingthe standard Phenol Chloroform Isoamyl alcohol (PCI) protocol. Specific set of primers were designed for the amplification of TPH2 gene exons and partial region of the introns. The amplified PCR products was precipitated and sequenced for the identification of variants. For sequence data analysis Chromas Software(2.1) was used along with BLAST available at NCBI and Clustal W program. Multiple alignments were performed for polymorphism identification and association of identified polymorphisms was performed using SPSS.The significant association of the variant rs7305155 with Major Depressive Disorder indicates that TPH2 has a role in disease etiology.Understanding the extent of the role different genes play in MDDmay help in tailoring medication. “Gene-Response to drug” association studies have also shown that patients with certain polymorphisms might respond better to certain class of drugs. It is useful to know which variants are prevalent in our population.
Availability: Items available for loan: UVAS Library [ Call number: 2939-T] (1).
Polymorphism Analysis Of Bovine Tumor Necrosis Factor Alpha Gene And Its Association With Mastitis In Nili Ravi Buffaloes
Material type: Book ; Literary form:
Publisher: 2017 Dissertation note: Various number of factors cause hindrance in the milk production potential of buffalos. Mastitis is the costly and most prevalent disease causing production losses of dairy herds in Pakistan and elsewhere in the world. Susceptibility and resistance to mastitis is complex trait influenced by genetic variation of animals. Among these immunity gene variations, the polymorphism in tumor necrosis factor alpha gene (TNF-α) play important role in immune response to virus. Polymorphism in TNF-α gene is associated with mastitis susceptibility and resistance. It would be a potential candidate gene for imparting resistance mastitis in dairy buffalos. Blood sample were taken from the 20 Nili Ravi buffalos having clinical and subclinical mastitis. Extraction of DNA was done from frozen blood after thawing, using organic extraction method & also kit method followed by DNA quantification (i.e. gel electrophoresis and nanodrop). Total 5 primers were designed using Primer3 bioinformatics tool. All these primers were optimized using different protocols and a set recipe was obtained for each primer. The amplification of DNA samples was done one by one using all these five primers on optimized protocol. The amplicons obtained were subjected to agarose gel electrophoresis to check whether we have the required product or not using 100 kb ladder and then amplicones were send for the sequencing.
The sequencing analysis of resulted amplicon sequence was done using Bioinformatics software Finch TV. Total of 6 mutations were found while 5 were same in all the samples whereas 6th mutation was found only in clinical samples. It is valuable in accomplishing genetic progress for resistance and to improve the immune response. This study will paved the way for animal breeder for selection of Nili Ravi mastitis resistant buffalos for breeding. TNF-α gene polymorphism based marker is now available for screening of resistant bulls as well.
Availability: Items available for loan: UVAS Library [ Call number: 2936-T] (1).