1.
Pcr-Based Diagnosis Of Canine Parvovirus In Dogs
by Farhan Towakal | Prof.Dr.Masoos Rabbani | Prof.Dr.Khushi Muhammad | Prof.Dr.Zafar | Faculty of Veterinary Sciences.
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Publisher: 2007Dissertation note: Canine parvovirosis, caused by a haemagglutinating canine parvovirus (CPV), one of the most important acute viral infectious diseases of pups, had been prevalent in the country. In the present study, 40 faecal samples, from clinically suspected cases of parvovirus diseased dogs and stray dogs were collected. The samples were diluted and centrifuged to collect the supernatant. Being a haemagglutinating (HA) virus; the pre-filtered supernatant from all suspected samples was checked for any HA activity using 1% washed chicken erythrocytes. Out of total of 40 samples, 30 samples were found HA positive. All the HA positive samples and samples found negative were processed for extraction of viral DNA with Genomic DNA purification kit. The net isolated DNA samples were subjected to Polymerase Chain Reaction (PCR) by using specific primers (developed against the variable genomic region of the capsid protein of the DNA) of the CPV. One of the selected primer pair designated as (37, 38) amplified the genornic region present in the CPV-2, the other primer pair designated as (39, 40) that amplified the region present in the CPV-2b.Optimization of PCR was done for the molecular level diagnosis of canine parvovirus and this technique was previously not available or standardized in local conditions of Pakistan. With the use of primer pair (37, 38) best results were found on following optimizing conditions like annealing temperature 55°C, primers concentration (reverse and forward) 25pmoles, Magnesium Chloride concentration 2mM, DNA(Template) volume Spl, Taq DNA polymerase(12.5U) and 2001iM of each dNTPs. For the primer pair (39,40) best results were found on following optimizing conditions like annealing temperature 5 5°C, Magnesium Chloride concentration 1.5mM, primers concentration (reverse and forwid) 20pmoles, DNA(Template) volume 6il, Taq DNA polymerase(12.5U), 2001.iM of each dNTPs. The PCR products were analyzed and compared for their banding pattern of 681bp and 427bp amplified by the primer pairs (37, 38) and (39, 40), respectively, against the standard DNA ladder (1 OObp) by using horizontal agarose gel electrophoresis. One of the HJA positive samples was not amplified by the PCR. The results of this study showed that the specificity of PCR reaction as compared to the HA test and the presence of the CPV-2 and 2b in Pakistan as the prevalent pathogenic strains. This study has been a first step for the molecular characterizationldiagnosis of canine parvovirus in local conditions of Pakistan. It is hoped that this study will pave the way for further advanced studies on this topic.
Availability: Items available for loan: UVAS Library [Call number: 0972,T] (1).
2.
Study On Molecular Diagnosis Of Canine Distemper Virus
by Muhammad Zubair Shabbir | Prof.Dr.Masood Rabbani | Prof.Dr.Khushi Muhammad | Prof.dr.Zafar | Faculty of Veterinary Sciences.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2008Dissertation note: Samples from fourty five dogs were submitted to the University diagnostic Laboratory, University of Veterinary and Animal Sciences, Lahore from January, 2007 to January 2008 for diagnosis of CDV infection. These dogs presented to referring veterinarians with clinical signs suspicious of CDV infection. Hematological examination (lymphocyte count) was carried out using K-EDTA anti-coagulant added whole blood and RT-PCR tests were performed using biological fluid samples that include plasma, nasal and conjunctival swabs. Only distemper positive dogs by RT-PCR were followed up for subsequent lymphocyte count and prognosis of distemper infection.
All the distemper positive dogs were lymphopenic but the degree of severity was variable as the samples were collected from dogs of different ages and phase of the disease. The study revealed that lymphopenia can be used to support presumptive clinical diagnosis but required laboratory procedure for confirmation and animal regain its normal value with the passage of time subjected to recovery. During followed up, two dogs were found to be dead because of CDV infection mixed with secondary bacterial infection in which one exhibited the nervous sign like teeth grinding, ataxia, convulsions and in coordination in body movements.
Only ten (22.22%) samples were found positive by RT-PCR using plasma, nasal and conjunctival swabs. CDV RNA was detected in 60% of plasma samples, 70% of nasal and 100% of conjunctival swab sample from lymphopenic dogs whereas the percentage was 13.33, 15,55, and 22.22 from a total of 45 samples. No amplicon of expected length was obtained from normal healthy dogs. On comparison of different fluid samples, the sensitivity of conjunctival swab was found to be highly significant followed by nasal swab and plasma.
In conclusion, Lymphopenia is the suggestive of clinical infection of dogs with canine distemper virus ad can help in presumptive diagnosis. It is not necessary that all lymphopenic dogs are distemper posit it requires further laboratory confirmtion. In this context, RT-PCR is test of choice with samples including conjunctival swabs and plasma.
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3.
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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4.
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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