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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.

Material type: book Book; Format: print 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: book Book; 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. Availability: Items available for loan: UVAS Library [Call number: 1034,T] (1).

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