Javed Muhammad (2010-VA-65)
Molecular And Serological Characterization Of Soilborne Francisella Tularensis - 2016. - 103p.;
Tularemia, caused by Francisella tularensis (F. tularensis ), is a zoonotic disease transmitted through contact with infected animals and contaminated environment. The disease has been reported from many countries of the world but no study has been done in Pakistan. In the current project, a total of 2280 soil samples representing 456 villages of eight districts of Punjab province were collected from way-points having human-animal interaction, processed for genomic extraction and tested through real time PCR for presence or absence of F. tularensis. Association of risk factors was determined from data such as gender and age of animals, plough method, irrigation system, fertilizer type used, availability of veterinary services, level of farmer education, physical and chemical composition of the soil. Moreover, sero-prevalence against F. tularensis in cattle, buffaloes, sheep and goats was determined using ELISA. Seventy four soil samples (3.24 percent) were found positive for F. tularensis. Phylogenetic analysis showed 100 percent similarity index with F. tularensis sub specie holarctica reported from other regions like USA, Sweden, Spain, Turkey and Germany. Presence of F. tularensis in soil showed negative association with increase in number of human density (0.7159; 0.3834-0.2054). Prevalence of anti- F. tularensis ELISA antibodies were significantly higher (p<0.05) in large ruminants (cattle and buffalo) as compared to small ruminants (goat and sheep). Age and gender-wise analyses showed non-significant differences (p>0.05) between small and large ruminants. Whereas, rain-irrigation system (2.96: 1.35- 6.48), lack of veterinary services (4.77:1.26-18.03) and use of organic fertilizer (5.3: 11.38- 20.39) have positive association with prevalence of anti- F. tularensis ELISA antibodies in the serum. Sero-prevalence of Ft in the animals has significant association with quantity of clay in soil (p<0.05). A conventional PCR based test has also been optimized for
Summary
103
detection of F. tularensis using tul4 gene specific primers. Specificity of primer showed F. tularensis detection in soil DNA in the presence of other cross-reactive organism. Sensitivity was determined in two fold dilutions with detection limit of up to 320 pg/μL. Utilizing pET28a vector, a construct was prepared containing transformed tul4 gene (450bp) showing 100 percent sequence homology to query gene sequence. For manufacturing diagnostic assays especially in developing countries where availability of BSL-3 facilities and positive control reagents is an issue, provision of tul4 gene based constructs in vector can act as positive control and biosafe to use.
It is recommended that similar studies may be done in other parts of Pakistan to have spatial distribution of F. tularensis all over Pakistan. In future studies, other sources of transmission like water, ticks and rodents may be considered with soil for complete analysis. Transportation of whole genome of F. tularensis has been prohibited by Russian government, ATCC and CDC, WHO is working on designing a complete protocol for transportation of this bacteria or genome to other countries. Under such situation, conventional PCR optimization can be done for diagnosis of F. tularensis and pET28+tul4 constructs, developed in this study, can be used as a PCR positive control reagents.
Microbiology
Phd. Theses
2640-T
Molecular And Serological Characterization Of Soilborne Francisella Tularensis - 2016. - 103p.;
Tularemia, caused by Francisella tularensis (F. tularensis ), is a zoonotic disease transmitted through contact with infected animals and contaminated environment. The disease has been reported from many countries of the world but no study has been done in Pakistan. In the current project, a total of 2280 soil samples representing 456 villages of eight districts of Punjab province were collected from way-points having human-animal interaction, processed for genomic extraction and tested through real time PCR for presence or absence of F. tularensis. Association of risk factors was determined from data such as gender and age of animals, plough method, irrigation system, fertilizer type used, availability of veterinary services, level of farmer education, physical and chemical composition of the soil. Moreover, sero-prevalence against F. tularensis in cattle, buffaloes, sheep and goats was determined using ELISA. Seventy four soil samples (3.24 percent) were found positive for F. tularensis. Phylogenetic analysis showed 100 percent similarity index with F. tularensis sub specie holarctica reported from other regions like USA, Sweden, Spain, Turkey and Germany. Presence of F. tularensis in soil showed negative association with increase in number of human density (0.7159; 0.3834-0.2054). Prevalence of anti- F. tularensis ELISA antibodies were significantly higher (p<0.05) in large ruminants (cattle and buffalo) as compared to small ruminants (goat and sheep). Age and gender-wise analyses showed non-significant differences (p>0.05) between small and large ruminants. Whereas, rain-irrigation system (2.96: 1.35- 6.48), lack of veterinary services (4.77:1.26-18.03) and use of organic fertilizer (5.3: 11.38- 20.39) have positive association with prevalence of anti- F. tularensis ELISA antibodies in the serum. Sero-prevalence of Ft in the animals has significant association with quantity of clay in soil (p<0.05). A conventional PCR based test has also been optimized for
Summary
103
detection of F. tularensis using tul4 gene specific primers. Specificity of primer showed F. tularensis detection in soil DNA in the presence of other cross-reactive organism. Sensitivity was determined in two fold dilutions with detection limit of up to 320 pg/μL. Utilizing pET28a vector, a construct was prepared containing transformed tul4 gene (450bp) showing 100 percent sequence homology to query gene sequence. For manufacturing diagnostic assays especially in developing countries where availability of BSL-3 facilities and positive control reagents is an issue, provision of tul4 gene based constructs in vector can act as positive control and biosafe to use.
It is recommended that similar studies may be done in other parts of Pakistan to have spatial distribution of F. tularensis all over Pakistan. In future studies, other sources of transmission like water, ticks and rodents may be considered with soil for complete analysis. Transportation of whole genome of F. tularensis has been prohibited by Russian government, ATCC and CDC, WHO is working on designing a complete protocol for transportation of this bacteria or genome to other countries. Under such situation, conventional PCR optimization can be done for diagnosis of F. tularensis and pET28+tul4 constructs, developed in this study, can be used as a PCR positive control reagents.
Microbiology
Phd. Theses
2640-T