1.
Molecular Detection Of Babesia Bigemina And Babesia Bovis In Carrier Cattle By Duplex Polymerase Chain Reaction
by Muhammad Suleman | Prof. Dr. Zafar Iqbal Ch | Dr.Asim Aslam | Prof. Dr Abdul Rauf Shakoori | Faculty of Veterinary Sciences.
Material type: ; Format:
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not fiction
Publisher: 2006Dissertation note: Babesiosis is a highly important disease in the world, caused by the intraerythrocytic protozoan parasites of the genus Babesia. A wide range of domestic and wild animals and occasionally man are affected by this disease, which is transmitted by ticks and has a worldwide epidemiological distribution. While the major economic impact of babesiosis is on the cattle industry, infections also occurs in other domestic animals , including horses, sheep, goats, pigs and dogs.
The present study targeted the carrier cattle infected with Babesia bigemina and Babesia bovis, as they are difficult to detect because of the low numbers of parasites that occur in peripheral blood. However, diagnosis of low-level infections with the parasite is important for studying the transmission and standardizing epidemiological studies. Using the Polymerase Chain Reaction (PCR) to amplify a portion of the gene from the parasite, and tested the ability of this method to detect carrier cattle.
A study was conducted to detect the. Babesia in blood samples through PCR based techniques. A PCR assay was described which could differentiate Babesia bigemina and Babesia bovis by using specific primer in carrier cattle. Blood samples of 100 cattle were randomly analyzed with PCR assay 29 (29.0%) out of 100 blood samples were positive for babesiosis in which 18% were positive for Babesia bigemina and 11% were positive for Babesia bovis, While the Light Microscopy detected only 18 (18%) out of the same samples. The samples found positive by LM were reconfirmed during the PCR assay but no sample was found to be having both Babesia bigemina and Babesia bovis infections simultaneously.
Thus it is concluded that PCR is a reliable molecular diagnostic technique to detect low level of infections in carrier animals in a population and thus could be used as an effective screening tool for the control and eradication of disease.
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2.
Standardization Of Avian Leukosis Diagnostic Techniques Through Polymerase Chain Reaction (Pcr) And Confirmation With Enzyme Linked Immunosorbant Assay (Elisa)
by Abdul Razzaq (M.Phil) | Prof. Dr. Zafar Iqbal Ch | Mr. Asim Aslam | Prof. Dr.Abdul Rauf Shakoori | Faculty of Veterinary Sciences.
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print
Publisher: 2006Dissertation note: Avian Leukosis Virus type J infection of chickens is a neoplastic disease affecting chickens. ALV-J is of great economic significance not only because of tumor mortality, but also because of decreased egg production in meat breeding stocks, increased rate of infections, poor response to vaccination and weight suppression in broilers. There is wide spread prevalence of ALV-A and ALV-J in commercial chicken flocks. For control of ALV's eradication programmes based solely on dam testing may be less effective than those where dam testing is combined with procedures to mitigate early horizontal transmission in progeny chicks. For this purpose PCR along with antigen capture ELISA was used in combination for detection of ALV-J proviral DNA, and ALV group specific antigen i.e. p 27 antigen of ALV-J. Polymerase chain reaction technique was standardized by using improved version of H7 primers specific for ALV sub group J targeting env gene encoding gp85 for the detection of avian leucosis virus type J and its confirmation was carried out by comparing it with antigen capture immunosorbant assay which measures group-specific antigen (GSA) i.e. p27 antigen. Feather pulp and serum samples from 50 broiler birds of up to 7 weeks of age were randomly selected from 10 different broiler poultry farms of district Lahore Pakistan. The prevalence of ALV-J was 22 % for antigen capture immunosorbant ELISA and 34 % for polymerase chain reaction (PCR).
Availability: Items available for loan: UVAS Library [Call number: 0943,T] (1).
3.
Detection Of Mycobacterium Tuberculosis And Mycobacterium Bovis From Spum And Blood Samples Of Human Using a Duplex PCR
by Asma Nawaz | Prof.Dr.Zafar Iqbal Ch | Dr.Azhar | Dr.Muhammad Younas Rana | Faculty of Veterinary Sciences.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2009Dissertation note: Tuberculosis is common infectious disease in the world. Mycobacterium tuberculosis is the most common cause of tuberculosis in the humans. Tuberculosis is endemic in Pakistan with about 1.5 million people infected. M.bovis is the major cause of gastrointestinal tuberculosis in humans.
The study was conducted in Lahore to compare 100 blood and 100 sputum samples from patients of active tuberculosis. The methods employed were conventional methods including Ziehl-Neelsen staining, culture on Lowenstein Jenson medium and biochemical tests. The Duplex PCR and conventional methods for diagnosis of tuberculosis caused by M.bovis and M.tuberculosis were compared on the sputum and blood samples. For M.tuberculosis and M.bovis the pncA gene and the species-specific 500-bp fragments were targeted in the Duplex PCR, respectively.
The sensitivity and specificity of Duplex PCR was found statistically significant in comparison to the conventional methods including Ziehl-Neelsen staining and culture for the differential diagnosis of tuberculosis caused by M.tuberculosis and M.bovis. Therefore Duplex PCR is a better choice of diagnostic test in the clinical setups where clinical urgencies necessitate a reliable, sensitive and specific test with the results in a short time period.
Availability: Items available for loan: UVAS Library [Call number: 1054,T] (1).
4.
Comparison Of Different Diagnostic Techniques For John'S Disease In Small Ruminants
by Saba Badar | Prof.Dr.Zafar Iqbal Ch | Dr. Mansur-ud-Din | Dr.Asim Aslam | Faculty of Veterinary Sciences.
Material type: ; Format:
print
Publisher: 2010Dissertation note: Paratuberculosis is one of the most hazardous infectious diseases, causing heavy economic losses due to poor health, low productivity and high fatality rate among domestic and wild ruminants. Mycobacterium avium subsp. paratuberculosis is the etiological agent of Bovine Johne's disease. In this study PCR were used to detect the presence of the Acid Fast Bacillus Mycobacterium paratuberculosis, in the intestinal tissues and Mesenteric Lymph Nodes of small ruminants causing Paratuberculosis. PCR was compared to HEY medium culture on the Herrold's Egg Yolk Media.
The samples were collected from Lahore Slaughter house and brought to the Molecular Pathology Laboratory at the Department of Pathology, University of Veterinary and Animal Sciences, Lahore. The study was conducted to compare PCR and the HEY medium culture for the diagnosis of paratuberculosis caused by M avium subsp. Paratuberculosis. A total of 500 tissue samples, 250 of the ileum and 250 of the mesenteric lymph nodes were collected randomly for the identification of Johne's disease. All samples were inoculated on the HEY medium prepared in the same laboratory aseptically. Followed by DNA extraction through the Kit method then run the PCR for insertion sequence IS 900, specific 626 bp fragment, were targetted in the genome of M paratuberculosis.
The results of the study showed more samples detected positive by PCR as compared to conventional culture methodology. Also they showed in the mass of 500 tissue samples that more bacilli are prone to the samples of small intestines than associated mesenteric lymph nodes. Regarding the sensitivity of the two techniques the PCR seemed more sensitive to detect the mycobacterium in the tissues than the conventional, laborious and time consuming HEY medium culture technique; though culture has been used as golden standard in this study also. When statistically analyzed results were insignificant due to small sample size.
The study will help in comparison of the two latest techniques for the diagnosis of M paratuberculosis, to check the validity of the better technique. In this study the sensitivity and specificity of PCR was checked and compared with culture on the HEY medium staining for the diagnosis of paratuberculosis in small ruminants.
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5.
Immunohistochemical And Pathomorphological Studies Of Chronic Granulomatous Enteritis (John'S Disease) in Bovines
by Muhammad Shahid | Prof.Dr.Zafar Iqbal Ch | Dr.Asim Aslam | Prof.Dr.Khushi Muhammad | Faculty of Veterinary Sciences.
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Publisher: 2010Dissertation note: Paratuberculosis, a disease caused by Mycobacterium paratuberculosis is a peril for both livestock and human beings. The present project was designed to study the pathmorphological changes induced by the organism and standardize more reliable diagnostic techniques to identify the M paratuberculosis. Tissue samples from ileurn and mesenteric lymph nodes were randomly collected from 1 50 cattle and buffalo, each in present study that was conducted in Lahore.
Gross lesions were recorded on a Performa. The samples were subjected to acid fast staining of smears from pellets after density gradient centrifugation and paraffin embedded tissue sections. All the samples also subjected to polymerase chain reaction and immunohistochemistry. The smears prepared from bacterial pellets of mucosal and cortical scraping of terminal ileum and MLN were stained indicated 11.4 % small intestine and 12.7% lymph nodes of cattle's and 8.7% and 10.7% lymph nodes of buffalo's tissue samples were positive. ZN staining of paraffin embedded tissue showed 8.0 % small intestine and 10% MLN of cattle's and 6.0 % of small intestine and 8.7% MLN in buffalo's tissue samples were positive. On basis of PCR 5.4% intestinal tissue samples and 6.0% MLN of cattle were positive. 3.4% intestinal tissue samples and 07(4.7%) MLN of buffaloes were positive. In buffaloes 4.0% intestinal tissue samples and 6.0% MLN were positive by IHC. In cattle 6.7% intestinal tissue samples and 8.0% MLN tissue samples were positive by IHC. In cattle, 27/150(18.0%) animals showed lesions in both intestine and mesenteric lymph nodes while 5/32 (15.7%) animals showed lesions in lymph nodes only. Out of 27/150(18.0%) intestinal tissue samples, 20/27 (74.1%) samples showed corrugation of the intestinal mucosa while 7/27 (26%) showed diffuse thickness.
In buffalo, 24/150 (16.0%) animals showed lesion in both intestine and mesenteric lymph nodes while 2/26 (7.7%) animals showed lesion in lymph nodes only. Out of 24 intestinal tissue samples, 19/24(79.2%) with gross lesion, samples showed corrugation of the intestinal mucosa while 5/24(20.9%) showed diffuse thickness.
In histopathology 20/27 samples of cattle showed focal granulomatous lesions while 7/27(26%) samples showed sever infiltration of macrophages and lymphocytes while 28/32(87.5%) lymph nodes showed infiltration of paracortical and cortical region of the lymph nodes with macrophages ,lymphocytes and multinucleated giant cells While 4/32 (12.5%) samples showed moderate infiltration of macrophages.
In buffaloes 19/24 (12.7%) samples showed focal granulomatous lesions while 5/24 (20.9%) samples showed sever infiltration of macrophages and lymphocytes while 22/26 (84.7%) lymph nodes showed infiltration of paracortical and cortical region of the lymph nodes with macrophages ,lymphocytes and multinucleated giant cells While 4/26 (15.4%) samples showed moderate infiltration of macrophages.
The sensitivity and specificity of immunohistochemical method was found significant in comparison Ziehl-Neelsen staining and histopathology for the diagnosis of paratuberculosis in cattle and buffaloes.
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6.
Polymerase Chain Reaction And Restriction Fragment Length Polymorphism (Rflp) By Using Ssu-r DNA Amplification for the Species Specific Diagnosis of Trypanosomiasis in Horses
by Naveed Sabir | Prof.Dr.Zafar Iqbal Ch | Dr.Asim Aslam | Prof.Dr.Khushi Muhammad | Faculty of Veterinary Sciences.
Material type: ; Format:
print
Publisher: 2010Dissertation note: In the current research project, a pari-trypanosome polymerase chain reaction (PCR) was optimized by using 18S single sub unit ribosomal DNA amplification and restriction fragment length polymorphism (RFLP) was also optimized and evaluated for the species specific diagnosis of the trypanosomiasis in horses.
Blood samples from one hundred (100) suspected horses were collected aseptically from different localities of Lahore. Fresh blood smear was prepared from each sample. After drying and fixing with absolute methanol, the slides were stained with Giemsa stain. Microscopic examination of stained blood smears revealed 8 positive samples out of one hundred (100) suspected horses. Polymerase chain reaction (PCR) was carried out on the same trypanosomiasis suspected blood samples to evaluate its sensitivity. Genomic DNA was extracted by using Genomic DNA Purification Kit (Fermentas mci., USA). The PCR was performed in a 50 tl reaction mixture. The tubes containing PCR mix were subjected to amplification cycles in a thermocycier after adjusting the amplification conditions.
After completion of the amplification cycles, the PCR product was characterized by 1.2 % agarose gel electrophoresis along with 100 bp DNA ladder to estimate the size of the PCR product and the gel was photographed with a Polaroid camera. PCR gave a higher percentage of positive cases i.e. 21% as compared to microscopic examination. Semi-nested polymerase chain reaction was carried out on product of the first run amplification by using same reaction mixture and amplification conditions except for template DNA. In case of semi-nested PCR 1 tl of the simple PCR product was used. Semi-nested PCR gave 100% (21/21) results. Restriction fragment length polymorphism (RFLP) analysis was conducted on nested products of the positive samples. A reaction mixture of 20 1iJ was used and samples were incubated over night at 37 °C in an incubator. The restricted products were characterized by 2 % agarose gel electrophoresis along with 100 bp DNA ladder and photographed with Polaroid camera. Restriction fragment length polymorphism (RFLP) analysis of the nested products revealed that none of the species including T. congolense, T. theileri, T. brucei and T. vivax was found in all (2 1%) positive animals having trypanosoma infestation.
It can be concluded from current study that a pan-trypanosome polymerase chain reaction is a superior and sensitive test as compared to Giemsa stained blood smear examination. The test can not only be used for early diagnosis of the trypanosomiasis but it can also be used to screen out the carrier animals those act as a reservoir of the infection for the horses and other susceptible animals. The advantage of this test is its sensitivity, universal applicability and the existence various possibilities for restriction enzyme analysis of the amplified region depending on the trypanosome species.
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7.
Tissue Residue Studies Of Enrofloxacin In Broilers Chicks
by Irfan Irshad | Prof.Dr.Zafar Iqbal Ch | Dr.Asim Aslam | Prof.Dr.Muhammad Athar Khan | Faculty of Veterinary Sciences.
Material type: ; Format:
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Publisher: 2009Dissertation note: Poultry industry is the second largest industry of Pakistan. Antibiotics are enormously used in poultry both for prophylactic and treatment purpose. Irrational use of antibiotics in poultry industry has led to the serious concern among the general public. It has also resulted in emergence of drug resistance in many susceptible organisms. The present study has therefore been planned for quantitative detection of Enrofloxacin residues; in tissues (liver, kidney, fatty tissues and muscles) of broiler birds. So, the present study was designed to detect Enrofloxacin residues in tissues of birds reared under experimental conditions and routinely slaughtered at different poultry shops. The study was completed in two phases. In phase-I, tissue samples from 75 broiler birds reared at Department of Pathology, University of Veterinary and Animal Sciences, Lahore were analyzed for quantitative detection of Enrofloxacin by HPLC. In phase-TI, the 25 broiler birds were purchased from various poultry shops of different local markets of Lahore. Tissue sample (Liver, Kidney and thigh muscles) from these broiler birds were also analyzed for quantitative detection of Enrofloxacin by HPLC. In experimentally reared birds, the highest concentration of Enrofloxacin observed was 306ng/g. In the birds injected with Enrofloxacin intramuscularly the overall highest concentration was 68ng/g. The concentration in kidney, liver and thigh muscles was in the range of 28-64 ng/g, 26-63 ng/g, 26-68ng/g in kidney, liver and thigh muscles respectively in birds injected with drug intramuscularly. The drug residues were detected up to 120 hours post treatment in intramuscularly injected birds. In orally treated birds level of Enrofloxacin in the kidney, liver and muscles were between 56-2 17 ng/g, 29- 306 ng/g ,27- 170 ng/g. The residues were detected up to 96 hours post treatment in birds given Enrofloxacin orally. The result of phase-Il showed that among the 75 market samples, 10 (40%) muscles, 8 (32%) liver and 7 (28%) kidney samples showed the Enrofloxacin residues. Out of 25 samples in which Enrofloxacin residues were detected 20 (80%) samples showed the residues concentration above MRL.
This study helped us in drawing true picture about Enrofloxacin drug residues in poultry meat and it is clearly indicated that proper withdrawal time is not being observed while marketing birds. This poses a great health concern for end consumer.
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8.
Study Of Pathogenesis Of Mycoplasma Gallisepticum In White Leg Horn Layer
by Mubasher Rauf | Prof.Dr.Zafar Iqbal Ch | Dr Aftab | Dr.M.Younus Rana.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2010Dissertation note: In first part of present study 380 samples were collected from clinically suspected cases of layers suffering from respiratory diseases in and around Lahore. Samples were subjected for mycoplasma isolation by using Frey's medium. Plates with positive growth revealed characteristic colonies on 8th day post inoculation that reached maximum in size and growth at 15th day post inoculation. Out of 380 samples, 104 (27.36%) samples were positive on culture. Isolates were identified through growth inhibition test (GIT) by using hyper immune sera raised in rabbits. Isolates were further confirmed by PCR. Similarly, tracheal swabs and tissue samples of lungs and trachea collected under refrigeration were also subjected for DNA analysis. Out of 380 samples 264 (69.5%) were positive on PCR analysis. By comparing two diagnostic techniques it was found that PCR was more sensitive and reliable technique for screening of Mycoplasma gallisepticum.
In second part of study experiment isolates were analyzed for protein profile of Mycoplasma by standardization of two techniques Sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) and western blotting. These techniques help to find out any antigenic variation in prevailing strain of mycoplasma. During our study five bands of protein were detected with molecular size of 32.35 kDa, 43.65 kDa, 52.48 kDa, 64.56 kDa and 70.8 kDa. These proteins were extracted from whole cell of Mycoplasma gallisepticum isolates. On comparing the molecular sizes it was found that isolated species showed low antigenic variation, analyzed by SDS-PAGE. Western blot was used to determine the specific protein of Mycoplasma gallisepticum with the use of specific polyclonal antibody raised in rabbit. The positive reaction site was shown on nitrocellulose membrane confirming target species of CRD.
During third part of present study, it was concluded that aerosol route of infection causes early disease, followed by intra tracheal and per-oral route respectively. The severity of infection was found more in aerosol and intra tracheal routes of inoculation than per-oral route which was found to be very mild. The general gross lesions observed in the above two groups were hemorrhages in trachea with mucous plug. There was air sacculitis, hemorrhages in the lungs, salpingitis and putrefied eggs in the ovary. On histopathological examination lesions were found in trachea, lungs and oviduct.
Re-isolation was carried out to confirm antigen in experimentally inoculated birds. Paraffin embedded sections of trachea, lungs and oviduct were processed for immunohistochemical examination in order to confirm the antigen of Mycoplasma gallisepticum within tissue. A positive immunochemical reaction was found in lungs and oviduct. Which represents that antigen was same as inoculated during study of pathogenesis.
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