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1. Isolation Of Local Strain Of Toxoplasma Gondii Through In-Vivo Cultivation In Mice

by Rahim Gul | Dr. Muhammad Imran Rashid | Dr. Aneela | Dr. Nisar Ahmad.

Material type: book Book; Format: print ; Literary form: not fiction Publisher: 2013Dissertation note: Toxoplasma gondii is an obligate apicomplexan, intracellular, parasite that infects all warm-blooded vertebrates, including mammals and birds. Human beings can be infected by ingestion of oocysts from cat faeces or through the consumption of meat containing Toxoplasma gondii cysts. Thus, food animals can be the source of transmission of Toxoplasmosis in human population especially among people who consume undercooked meat in the forms of barbecues, beef steaks, kebabs, burgers and shawarmas. Oocysts of T. gondii from cat faeces were identified by using direct microscopy and flotation technique. The positive oocysts were confirmed by micrometry having diameter of 9-13 ìm. The oocysts were then sporulated in aerated condition. After sporulation oocyst were inoculated in Swiss albino mice for in-vivo culturing. After 56-70 days brain tissue was collected from infected mice and subjected to DNA extraction and PCR amplification. Similarly DNA was also extracted from sporulated oocyst for copro-PCR. Out of 200 faecal samples only three were found positive for Toxoplasma gondii through direct microscopic examination and flotation technique. From positive faecal sample and brain tissue DNA was extracted by QIAGEN mini stool kit and QIAGEN DNA mini kit. After DNA extraction the samples were examined through PCR by using specific Toxoplasma gondii B1 gene primer having 529 bp size. Two hundred faecal samples were examined for T. gondii using direct microscopy, flotation technique, bioassay and polymerase chain reaction. Out of 200 samples 3 (1.5%) were found infected through direct microscopy and flotation technique. Toxoplasmosis was more prevalent in adult cats (1.65%) as compared to young ones. Prevalence was also found high in females (2.08%) as compared to males. Similarly healthy cats have higher prevalence rate (1.30%) as compared to diseased ones. A further confirmation was done through polymerase chain reaction and brain tissue cyst Bioassay give 1 positive amplification while Copro-PCR gives 2 positive amplifications. Therefore it can be concluded that the copro-PCR is can be used for the confirmation of Toxoplasma oocysts from cat faeces and tissue cysts from bioassay in mice. Therefore, we propose that the copro-PCR can be used as the new gold standard for determining potential cat infectivity and tissue cysts from bioassayed mice or contaminated meat samples of livestock. Availability: Items available for loan: UVAS Library [Call number: 1778,T] (1).

2. Phylogenetic Analysis Of Haemoproteus In Chicken And Sparrows

by Anha fatima | Dr. Muhammad imran rashid | Dr. Azhar maqbool | Dr. Wasim.

Material type: book Book; Format: print ; Literary form: not fiction Publisher: 2014Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 2038,T] (1).

3. Development Of Molecular Tools For The Diagnosis Of Plasmodium Vivax Using Cytochrome C Oxidase Gene

by Ayaz Shaukat | Prof. Dr. Azhar Maqbool | Dr. Muhammad | Dr. Muhammad Imran Rashid.

Material type: book Book; Format: print Publisher: 2014Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 2153,T] (1).

4. Incidence Of Canine Trypanosomiasis And Standardization Of PCR For Its Diagnosis

by Sajid Bashir Khan Qaisrani (2006-VA-60) | Dr. Muhammad Haroon Akbar | Dr. Muhammad Imran Rashid | Dr.Wasim Shahzad | Faculty of Veterinary Sciences.

Material type: book Book; Literary form: not fiction Publisher: 2014Dissertation note: Thesis Submitted With Blank CD. Availability: Items available for loan: UVAS Library [Call number: 2198,T] (1).

5. Isolation Of Surface Antigen 1 Gene Of Toxoplasma Gondii And Its Cloning In The Expression Plasmid

by Farooq Riaz (2008-VA-231) | Dr. Muhammad Imran Rashid | Prof. Dr. Kamran Ashraf | Dr. Jawad Nazir.

Material type: book Book; Literary form: not fiction Publisher: 2015Dissertation note: Toxoplasma gondii is an obligate intracellular protozoan parasite which comes under the classification of phylum Apicomplexa, subclass Coccidiasina (Cornelissen et al. 1984). Toxoplasmosis is one of the more common parasitic zoonoses world-wide caused by Toxoplasma gondii which is a facultatively heteroxenous, polyxenous protozoon that has developed several potential routes of transmission within and between different host species (Tenter et al. 2000). It is the most important source of toxoplasmosis in humans and animals, with cat as definite host and warm-blooded animals as intermediate host (Frenkel et al. 1970). It was first described by Nicolle, Manceaux and Splendore in 1908 from rodents Ctenodactylus gondii (Black and Boothroyd 2000). Toxoplasmosis is a worldwide parasitic disease and it is estimated that about one-third total population of the world is seropositive for Toxoplasma gondii (Tenter et al. 2000). Prevalence of infection varies between countries, geographical areas and ethnic groups living within a specific region. In Humans, infection rates range from 50% to 83% in Brazil (Tenter et al. 2000; Dubey et al. 2012). Seropositivity of Toxoplasma gondii in China is about 8% with continuously increase while in USA its 10-15%, 50-70% in France and 20% in UK (Dubey and Jones 2008; Zhou et al. 2008; Jones et al. 2009). Prevalence of toxoplasmosis is higher in males (79%) as compared to females (63.4%) and the age dependent sero-prevalence reaches >92% in age group of 40 to 50 (Coêlho et al. 2003). Transmission occurs through the ingestion of contaminated vegetable /water with oocysts, as well as the ingestion of contaminated raw/undercooked meat with tissue cysts (Gajadhar et al. 2006). Transmission may also occurs by ingestion of sporulated oocysts, or bradyzoites within cysts present in the tissues of numerous food animals (Esteban-Redondo et al. 1999). In humans, transmission of Toxoplasma gondii happens mainly by eating raw or undercooked contaminated meat, raw cow’s milk and birds eggs, swallowing oocysts dis-charged in feces of infected cats, inoculation of trophozoites through the skin, or by inhalation (Wallace 1971; Wallace 1973; Bannister 1982). In humans, mostly infections (congenitally or post-natally acquired) are asymptomatic. Congenital infection occurs only when a woman becomes infected during pregnancy. Congenital infections acquired during the first trimester are more severe than those acquired in the second and third trimester (Desmonts and Couvreur 1974). The main clinical signs associated with toxoplasmosis are anorexia, weight loss, lethargy, dyspnea, ocular signs, pyrexia, vomiting and diarrhea, jaundice, myositis, encephalitis and abortion. Humans become infected when they ingest the toxoplasma at infective stages (oocysts and tissue cysts) found in some cat feces and in raw meats. In addition to being hazardous to livestock animals, the T. gondii infection is also important due to its zoonotic implications (Jittapalapong et al. 2005). Congenital abnormalities in humans, such as microcephaly, hydrocephaly, chorioretinitis, convulsion, cerebral calcification, epilepsy, blindness, deafness, and mental retardation may occur if the mother acquires infection during pregnancy (Jones et al. 2003). In addition to congenital anomalies, T. gondii also causes severe neuropathologic infections in immuno-compromised hosts, such as AIDS and cancer patients receiving chemotherapy (Del Valle and Piña-Oviedo 2005). Seroprevalence studies of T. gondii among domestic animals in South-Western Pakistan has indicated considerable prevalence (25% in cattle, 2.5% sheep) (Zaki 1995) and suggesting potential transmission to the human community. Small scale study in urban area of Rahim Yar Khan (Punjab), Pakistan has revealed that the overall prevalence of toxoplasmosis in food animals is 19% (Ramzan et al. 2009). Another study has already been published that untreated patients with leprosy in Pakistan have shown significant seroprevalence (29.6%) of antibodies against T. gondii (Hussain et al. 1992). Vaccine against toxoplasmosis is not available yet with one exception (“Toxovax” for sheep). Vaccine against T. gondii in animals used for human consumption may block the possible transmission to humans (Bhopale 2003). SAG1, among one of the major antigenic components of Toxoplasma gondii is a major surface antigen identified on the surface membrane of this parasite using a monoclonal antibody (Handman et al. 1980). SAG1 is an important surface antigen, expressed by tachyzoite form of T. gondii and is a putative candidate for vaccine and diagnostic against toxoplasmosis (Sharma et al. 1983; Godard et al. 1990). Immunization with SAG1 adjuvanted with saponin Quil A or incorporated in lysosomes provided total protection after challenge (Bülow and Boothroyd 1991; Khan et al. 1991). SAG1 is single copy gene with no introns (Burg et al. 1988), regulates both humoral as well as cellular Th1 immune responses (Liu et al. 2008) and is powerful candidate for vaccine against toxoplasmosis. SAG1 is a potent candidate of diagnostics for detection of serum antibodies against toxoplasmosis in Man and animals (Abu-Zeid 2002). Availability: Items available for loan: UVAS Library [Call number: 2258-T] (1).

6. Molecular Diagnosis Of Anaplasmosis In Buffaloes

by Muhammad Salman (2008-VA-135) | Prof. Dr. Khalid Saeed | Dr. Muhammad Imran Rashid | Dr. Wasim Shehzad.

Material type: book Book; Literary form: not fiction Publisher: 2015Dissertation note: Bovine Anaplasmosis is a tick-borne haemo-rickettsail disease, caused by Anaplasma species transmitted mechanically by flies, biologically by ticks and blood contaminant fomites. It is an economically important tick-borne disease of buffalo in tropical and sub-tropical areas of the world. In current study, we developed and optimized PCR first for detecting Anaplasma at genus level in buffaloes. One hundred (100) blood samples were collected from buffaloes around the Lahore region. The stained thin blood films were examined microscopically and 37% blood samples were found positive for intra-erythrocytic bodies which were then selected for DNA extraction. The DNA was extracted using commercially available kit for eventual use in optimization of PCR for diagnosis of bovine Anaplasmosis. The primers were designed targeting 16S rRNA gene of Anaplasma. For the detection, the PCR product was run in 2% agarose gel stained with ethidium bromide and thirty seven samples showed the amplification band at 179bp. The selected samples were sent for ABI sequencing to Singapore for the accurate detection of the Anaplasma species. The sequencing results were blasted with database of Genbank and we observed homology with Anaplasma phagocytophilum. We found 37% prevalence of Anaplasmosis in buffaloes through PCR. However more studies are required to confirm the species of Anaplasma infecting buffaloes (Bobalus bobalis) by designing species specific primers. Furthermore, additional studies are needed to establish the epidemiology of Anaplasmosis by using molecular tools in different geographical areas of the country for their better control. Availability: Items available for loan: UVAS Library [Call number: 2389-T] (1).

7. Indigenous Elisa Kit For Toxoplasma Gondii: Optimization Of Antibody Detection Elisa Of Sag 1 Protein As An Antigen In Mouse Model

by Madiha Sana (2013-VA-957) | Dr. Muhammad Imran Rashid | Dr. Haroon Akbar | Dr. Wasim Shehzad.

Material type: book Book; Literary form: not fiction Publisher: 2015Dissertation note: Toxoplasma is an apicomplexan intracellular parasite which is the cause of toxoplasmosis in man and animals. It occurs by the ingestion of oocyst from feces of cats or by eating raw meat in which cysts are present. It is the one of the major cause of encephalitis and abortion in immuno-compromised animals and humans. As it is difficult to screen out infected live animals from field, it is important to vaccine animals as well as humans for toxoplasma to prevent its transmission from animals to humans and from humans to their off springs. Cloning of surface antigen genes plays an important role in development of vaccine and for serology of T. gondii. Enzyme linked immuno-sorbant assay proves to be a significant tool to estimate the humoral response elicited against expressed recombinant protein in mice. The recombinant protein of SAG1 was collected from Molecular Parasitology Laboratory, University of Veterinary and Animal Sciences, Lahore. In the previous studies, SAG1 sequence was cloned in the expression plasmid and successfully expressed in prokaryotic expression system. In the current study, rSAG1 was quantified by using BCA protein assay through BioWORLD protein quantification kit. In another experiment, the Swiss mice were immunized with 15 μg rSAG1 protein 3 times with 2 weeks intervals. Two groups of mice were formed with five mice in each group. Sera were collected after 2 weeks of each inoculation. For performing ELISA, four different experiments were performed with different concentrations i.e. 5μg/ml, 250μg/ml and 500μg/ml with two different dilutions; 1/50 and 1/20. The O.D. values of concentrations 5μg/ml and 250 μg/ml with two dilution series of 1/20 and1/50 were not observed significant while the antigen coating concentration of 500 μg/ml with 1/50 dilution showed 1:160 titre and with 1/20 dilution showed 1: 1280 titre after the 3rd shot. The O.D values with 500 CHAPTER 6 SUMMARY SUMMARY 36 μg/ml concentration with 1/20 dilution after the 3rd shot were observed significant in the inoculated group as compared to the O.D values of un-inoculated negative group. It is suggested to carry out ELISA with purified rSAG-1 protein and to optimize ELISA to test toxoplasma infected mice. Availability: Items available for loan: UVAS Library [Call number: 2433-T] (1).

8. Occurrence And Economic Losses From Theileriosis On Commercial Dairy Farm Of Holstein Friesian

by Muhammad Rashid (2014-VA-503) | Dr. Muhammad Imran Rashid | Prof. Dr. Khalid Saeed | Dr. Liaquat Ahmad.

Material type: book Book; Literary form: not fiction Publisher: 2016Dissertation note: Background: Theileriosis is a tick-borne disease and it is transmitted by the bite of ticks. Previous work on disease problems in the study area suggested that Ticks and Tick-Borne Diseases (TTBDs) are the major constraints to cattle production. They cause economic losses to farmers in terms of cattle mortality, loss of body weight, loss of milk production and costs of control of TTBDs by use of acaricides. Theileria is one of the major threat to cattle as it causes anemia, weight loss, decrease production, mortality, treatment cost and cost for the control of theileria. The proper data for losses atributed to theileriosis is still not available in Pakistan. For this purpose a study was carried out in a commercial exotic dairy farm to evaluate losses associated with theileriosis Methodology: The study was done during the period of theileriosis to calculate its economic effect on animal health and production. A total of 150 animals were selected randomly using random number sample formula. The animal tag numbers were compared with random number table, comparing animals were slecteded for study. Thin blood smear was performed for diagnosis haemoparasite, further PCR was performed on those animals that were found +ve for intraerythrocytic bodies. Faecal examination, California mastitis test, teat abnormality and parturition history were recorded for the screening of these factors that decrease milk production. After final grouping, milk production was recorded to identify the effect of theileriosis on production. As theileriosis cause anemia due to destruction of RBC’s. body condition scoring was also performed. Physical examination (lymph node and body temperature) of animals were also performed to evaluate the clinical and subclinical theileriosis. Results: For the evaluation of theileriosis, microscopy was performed on all the animals’ blood samples. Haemoparasites were found in 28.67%. These were further processed by PCR for the CHAPTER 6 SUMMARY Summary 55 detection of theileriosis. Theileria was found in 27.90%. Screening of clinical and subclinical mastitis by Califirnia Mastitis Test and microscopy for gastrointestinal parasite were performed. On faecal examination, there found nematode, cestode and balantidium in 51.72%, 60.92% and 42.53%% respectively. After deworming with Valbazine and curafluke, nematode, cestode (monzia), balantidium and coccidiosis were found in 0%, 39.13, 43.48% and 4.35% respectively. Before grouping clinical and subclinical mastitis were found in 5.38% and 24.62% respectively. After grouping clinical and subclinical mastitis were evaluated by California mastitis test with two weeks interval. At 7th week clinical and subclinical mastitis were 3.85% and 7.69% due to improved management. The decrease in milk production for clinical and subclinical theileriosis was 87 lit./animal and 42.77 lit./animal. Costs for control, treatment and mortality were 0.12%, 0.20% and 13.09% respectively from overall farm expenditure. The prevalence of haemoparasite was 28.67%, while the prevalence of theileriosis was 8%. The new cases of theileriosis were recorded and incidence of theileriosis was found to be 2.25%. Overall losses due to theileriosis was 13.70%. Outcomes: We can conclude from our finding that theileriosis has drastic affect on the profitability of the farms. Then losses can be attributed to decreased milk production and mortality. Medications and control measure for theileriosis have added effect on the losses at exotic animal breed dairy farms. Perspectives: Cost analysis studies need to be done on different dairy farms of cattle of different breeds at different ecological/climatic zones of Pakistan so that investors would know the risks of establishing dairy farms. Availability: Items available for loan: UVAS Library [Call number: 2515-T] (1).

9. Exploring Anthelmintic Resistance In Ovine Haemonchosis Through Faecal Egg Dna At Livestock Research And Development Station, Paharpur, D .I. Khan

by Ghulam Hassan (2007-VA-144) | Dr. Haroon Akbar | Dr. Muhammad Imran Rashid | Dr. Muhammad Ijaz.

Material type: book Book; Literary form: not fiction Publisher: 2016Dissertation note: Gastrointestinal nematodes are recognized as a major constraint of small ruminant production system at small and large-scale farming in developing countries, leading to significant economic losses. The most important of these is Haemonchus contortus. Anthelmintic resistance now poses problems to sheep farmers throughout the world. This study has been designed to check anthelmintic resistance against haemonchosis of sheep by an in vivo method. The current study was carried out at Parasitology laboratory (Toxovacc lab), Department of Parasitology, University of Veterinary and Animal Sciences, Lahore. 100 faecal samples were collected from sheep at Livestock Research and Development Station, Paharpur, D.I. Khan. Animals were drenched with anthelmintic (Albashell containing Albendazole 2.5%, administered @10 mg/kg of body weight) orally after 1st sampling, at 0 day. The faecal samples were examined microscopically, micrometery was exploited and EPG analysis was performed by using McMaster technique. After 14 days, the second sampling was done. The fecal samples were brought and stored at 4°C in Parasitology laboratory (Toxovacc lab). Pre-trial & Post trial EPG were compared and positive samples were taken (tag#1057 Damani sheep male, tag#13 Balkhi sheep male, tag#1096 Damani female, tag#06 Balkhi female, tag#20 Balkhi) for egg isolation (Module, 2004) for egg DNA extraction through classical method of Phenol-Chloroform-Iso-Amyl Alcohol extraction. DNA samples were subjected to polymerase chain reactions (PCR) targeting β tubulin gene for detection of benzimidazole resistance at genetic level. Fecal egg count reduction percentage of 74.57% at day 14 post treatment clearly shows the presence of benzimidazole drug resistance in parasites infecting Balkhi and Damani sheep at Livestock Research and Development Station, Paharpur, D.I. Khan. Summary 64 In conclusion, PCR-Sequencing technique finds its value in the detection of benzimedazole resistance at molecular level in eggs of Haemonchus contortus of sheep and this technique also helps the understanding of the development of drug resistance in the parasite. Availability: Items available for loan: UVAS Library [Call number: 2513-T] (1).

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