Isma Nazli Bashir

Molecular Detection And Speciation Of The Canme Piropiasm - 2008

An epidemiological study of babesiosis in dogs was conducted at Pet center, University of Veterinary and Animal Sciences, Lahore, for one year and information on age, sex and breed was gathered. It was found that from a total number of 6204, dogs up to two years of age were more susceptible than other age groups (2-4, 4-6 and above 6 years).The data regarding genders revealed that males were more prone to the disease than female dogs. As far as the breeds were concerned, crossbred dogs were more susceptible followed by Pointers, Alsatians, German shepherds and Bull terriors.Hot and humid months (June to September) have greater impact on the occurrence of disease. The study regarding identification of ticks revealed that Rhiphicephalus sanguinus is the predominant vector of the disease in Pakistan.
Molecular studies were conducted to characterize and identify the species responsible for canine babesiosis in Pakistan. In this regard, a nested polymerase chain reaction-Restriction fragment length polymorphism (PCR-RFLP) technique was employed on different specimens (Blood, Body tissues and Ticks). For this purpose blood samples were collected from twenty four chronically infected dogs and applied on the Flinders Technologies Associates (FTA) cards for transportation to Australia. Different body tissues (Liver, Spleen, Kidney, Intestine, Bone marrow and Pancreas) were procured after euthanizing the two dogs and DNA was extracted, for further studies. Similarly, the eighty eight ticks were also collected from the infested dogs in the 70% ethanol for transportation to Australia. A nested PCR-RFLP assay was used for the detection and differentiation of Piroplasm species on the basis of the 1 8S ribosomal RNA gene. The assay potentially amplified and identified Babesia gibsoni as the main canine piroplasm. Similar assays on the DNA extracted from body tissues and ticks revealed Babesia gibsoni as the main piroplasm. The PCR was found to have a high detection limit (equivalent to i0 dilution), when using the DNA extracted from blood applied to FTA cards, body tissues and ticks. A new technique was developed for extraction of DNA from FTA cards and tick, in this technique, instead of using the FTA specified punching machine, we used scalpel blades, and so the rest of the chemicals used are'generally and easily available. The same protocol was used for extraction of DNA from ticks, only chemicals used in different quantities with different spinning times. Both of which, resulted in cost reduction, less effort and speedy DNA extraction. The technique reported here has the potential to be standardized for routine DNA extractions from FTA cards and ticks.

Department of Parasitology
Phd. Theses


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