1.
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).
2.
Development Of Dna Based Diagnosis Of Babesia Canis In Dogs And Its Specificity With Peripheral Blood Smear Microscopy
by Muhammad Awais (2009-VA-539) | Dr. WasimShehzad | Dr. M. YasirZahoor | Dr. M. Imran Rashid.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Babesia canis (B. canis) is an intra-erythrocytic parasite and it is responsible for canine babesiosis. Mainly ticks are responsible for its transmission to vertebrate host. Currently, there are three sub-species of B. canis has been identified i.e. B. caniscanis(B. c. canis), B. canis vogeli (B. c. vogeli)and B. canis rossi (B. c. rossi).Clinical presentation and severity of infection are markedly different for each sub-specie. Routinely used methods for the diagnosis of canine babesiosis include clinical presentation, host/vector specificity, blood smear microscopy and Immunological testing (IFAT and ELISA).
In this study, blood smear microscopy and molecular based technique, PCR, were evaluated. This study has comparative as well as developmental nature.Peripheral blood smear microscopy is cost effective and quick method of diagnosis. But the limitations associated with microscopy include low parasitaemia in chronic and asymptomatic infection, morphological similarity of B. canis with other species of Plasmodium and Theileria. Furthermore, personal expertise in morphology based identification and slide preparation may also hamper the precise identification. These limitations may lead to misdiagnose the infection.
PCR based method, developed in this study, found to be more specific and sensitive than conventional microscopy. Fifty blood samples were collected from September, 2014 to November, 2014. These samples were screened microscopically as well as with PCR. B. canis-like bodies were identified in forty-two (42) samples. But all (50) samples were found positive for B. canis¬via PCR. Sequencing results following PCR have shown that identified sub-specie is similar withB. c. vogeli(99%).
This study clearly depict the likelihood of misdiagnosis with microscopy due to morphological similarities with other piroplasms. Therefore, PCR based methodology was found highly specific for B. canis and produced high throughput. Furthermore, sub-species differentiation is only possible with molecular based approach. As the described approach is found highly specific and sensitive than other conventional methods, therefore, it may aid in the development of clear canine babesiosis geographical distributional picture. Which ultimately help in the development of control and treatment strategies which may eradicate babesiosis.
Availability: Items available for loan: UVAS Library [Call number: 2287-T] (1).
3.
Seroprevalence And Molecular Detection Of Brucellosis In Hospitalized Patients With Clinical Manifestations Of Brucellosis
by Riffat Yousaf (2008-VA-342) | Dr. Wasim Shehzad | Prof. Dr.Tahir Yaqub | Dr. Haroon Akbar.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Brucella species are host specific facultative intracellular pathogens which cause brucellosis in both animals and humans. Brucellosis is one of the most common zoonotic diseases worldwide. In Pakistan, the incidence of brucellosis is increasing day by day due to lack of awareness of this deadly malady. It is transmitted from infected animals to humans who are in close contact with infected vaginal secretions, feces, blood, aborted fetus, or by consumption of unpasteurized milk and dairy products. Infection due to B. melitensis and B. abortus are mostly prevalent for brucellosis in human.
Total 218 blood samples were collected in gel vacutainer tubes from hospitalized patients who were clinically manifested with brucellosis. Out of 218, 12 RBPT positive blood samples were collected in EDTA containing vacutainer tubes separately. Serum was isolated from all blood samples (without EDTA). These serum samples were first screened by Rose Bengal Plate Test (RBPT). DNA was extracted from all positive RBPT blood and serum samples and randomly selected negative RBPT serum samples. All extracted DNA (≤10ng/µL) were subjected to Brucella genus and two species specific (B. abortus and B. melitensis) Quantitative Real Time Polymerase Chain Reaction (qRT-PCR) assay. Furthermore, few selected extracted DNA (≥20ng/µL) from blood and serum samples were examined by genus and Multiplex specie specific PCR. The PCR products were electrophoresed on 2.5% agarose gel. Then selected products were sequenced by ABI 3130 XL sequencer. The data were analyzed by SPSS software using Chi square test.
The present study helped to diagnose accurately and precisely brucellosis in clinical manifested patients, which is further helpful for devising the strategies to control this disease.
Availability: Items available for loan: UVAS Library [Call number: 2286-T] (1).
4.
Molecular Characterization Of Local Donkey Based On Mitochodrial D-Loop Analysis
by Shakeel Earnest (2012-VA-597) | Dr. Muhammad YasirZahoor | Prof. Dr. TahirYaqub | Mr. Tariq Mahmood .
Material type: ; Literary form:
not fiction
Publisher: 2014Dissertation 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).
