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Phase 4 (2009 Deadline)
Arthropod Functional Genomics Initiative: Building Community Resources for Animal Health
Shahid Karim, University of Southern Mississippi
Zafar Iqbal and Muhammad Sohail Sajid, University of Agriculture, Faisalabad
Pakistani Funding (Department of State): $206,146
US Funding (Department of State): $351,671
Project Dates on US Side: November 15, 2010 - November 14, 2013
Project Overview
In Pakistan, the livestock sector plays a key role in the country’s growth, representing more than 50 percent of agricultural output and more than 11 percent of the national gross domestic product. More than 90 percent of livestock is owned by small farmers and landless rural householders, who earn their livelihoods by selling milk and animals, but ticks and tick-borne diseases are a major limiting factor in animal health and wellbeing. Thus, effective tick control is vital to continued success of small livestock holders. The University of Southern Mississippi (USM) and the University of Agriculture Faisalabad (UAF) have formed a partnership to build a new molecular parasitology laboratory and training program at UAF aimed at developing diagnostic and preventive tools for vectors and vector-transmitted diseases. They will use genomic mapping to identify novel means of blocking tick feeding and pathogen transmission. This research project will provide valuable knowledge for future development strategies for tick control, as well as provide potential targets for drug and vaccine design aimed at blocking tick feeding on cattle. The broader impacts of this project will include not only a better understanding of prolonged arthropod vector feeding on vertebrate hosts but also enhanced training of faculty and future life scientists, who will gain valuable first-hand experience in the conduct of genomics research.
Quarterly Update
The identity of Rickettsia species associated with 574 ticks of 11 species collected from Pakistan was determined by sequencing the rickettsial outer membrane protein A (rompA) gene. The DNA sequence homology search revealed the presence of Rickettsia, Babesia, and Thelieria species associated with cattle disease in Pakistani ticks and have been catalogued. New species are being characterized. It should open up a new avenue of research in developing diagnostic tools, and their prevention (therapeutic/vaccine). The team identified over 5000 tick salivary genes using RNA-Seq technology. These tick salivary genes/proteins assist ticks in prolonged tick blood feeding on the host, and pathogen transmission. Identification of these salivary genes will assist in screening targets for anti-tick (anti-disease) vaccines and new pharmacologically active proteins (e.g. blood thinners, inhibitors of several blood problems etc). The visiting student, Asma Kausar completed her six month training at USM and returned to Pakistan in September 2013. Arrival of another graduate student, Mr. Jawad ul Hassan was delayed until October 6 due to administrative and visa issues. A third student, Ms. Quratul Ain, who is still waiting for her visa will bring additional samples from Pakistan and investigate the tick microbiome using functional metagenomic approach during her stay at the US lab.
Progress Reports
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2011 Show summary || Hide summary
By the end of 2011, approximately 2,000 ticks had been collected from various species (cattle, buffalos, poultry, dogs, camels, donkeys, goats, and sheep) from livestock farms in different ecological regions of Pakistan. All collected samples were shipped to the University of Southern Mississippi and have been identified to the species level. The National Tick Collection (University of Southern Georgia) has assigned accession numbers to all identified Pakistani tick species. Five hard tick species (Hyalomma anatolicum anatolicum, Hyalomma dromedarii, Rhipicephalus microplus, and R. sanguineus) were found on infested animals, and the soft tick species Argas persicus was also found, predominantly infesting Pakistani poultry. All tick DNA samples are being analyzed for the presence of tick-borne infections such as Spotted Fever Group Rickettsia (SFGR), Anaplasmosis, and Babesiosis.
Renovation work has been completed on the new Molecular Parasitology Lab at the University of Agriculture. New bench and desk spaces have been added to house new graduate students, research assistants, and postdoctoral fellows. New equipment (gel documentation system, gel electrophoresis systems, ultra low temperature freezer, -20ºC freezers, and bench-top refrigerated high-speed centrifuge) has been purchased and all installations should be complete by March 2012. A newly revised graduate-level course on Techniques in Molecular Parasitology is being taught by Pakistani co-PI Dr. Sajid, with six graduate students enrolled (four males and two females). Dr. Sajid plans on adding a lab component to his course, and newly purchased scientific equipment will be used to provide hands-on training to the enrolled students. Work has already commenced on establishing a pathogen-free Rhipicephalus microplus tick colony in the lab for use in functional genomics experiments. Meanwhile, project staff at UAF continue their outreach efforts to assist local farmers. Public events were held October 15 and December 11, 2011, to educate stakeholders on controlling disease-causing parasites affecting their livestock. In 2012 these researchers will continue their monthly webinar series to provide training to the Pakistani students on arthropod vector biology and tickborne diseases. Tick samples will continue to be collected and shipped under permit to Dr. Karim. Dr. Iqbal has also been invited to visit the University of Southern Mississippi, pending receipt of his visa, for which he has been waiting since his interview with consular staff in April 2011.
2012 Show summary || Hide summary
During this past quarter, Dr. Iqbal reports that about 500 tick samples were collected from various animals (cattle, buffalos, poultry, dogs, camels, donkeys, goats, and sheep) in Baluchistan and Kashmir. The collection efforts were facilitated thanks to linkages established with the University of Baluchistan in Quetta and a network of veterinarians in Kashmir. The samples have been shipped to the University of Southern Mississippi for identification and DNA extraction. After finally receiving his U.S. visa after a year of waiting, Dr. Iqbal arrived at USM on April 16, 2012, to receive functional genomics training and participate in analysis of the samples during his stay through June 8.
Meanwhile, back at UAF, two new graduate students (Asma Kausar and Jawad-ul-Hassan) have joined the Molecular Parasitology Lab. Photos of the newly remodeled and upgraded lab recently provided by Dr. Iqbal are included below. A new graduate course on Physiology of Parasites is also being offered from March through July 2012, with an emphasis on tick physiology. The UAF researchers have reported some difficulties associated with the unreliable electrical power supply in their area, and they are looking into the possibility of obtaining funds from their university to buy a generator. Although all the equipment expected to be purchased under their S&T grant has been purchased and installed, they have encountered some delays with their university’s lengthy procurement process for buying some of the reagents and supplies. Nevertheless, their collection and sample processing efforts are continuing, as are the monthly webinars in which the U.S. PI Dr. Karim is providing training to UAF students on arthropod vector biology and tickborne diseases. After Dr. Iqbal returns home to Pakistan in June, he and his co-PI Dr. Sajid will be organizing a hands-on training workshop on the use of molecular techniques in the diagnosis of tickborne pathogens. An international conference on parasitic threats to livestock is also planned for November 2012, and details on both events will be provided in subsequent reports.
Dr. Iqbal visited the University of Southern Mississippi (USM) for two months of training and research collaboration from April 13 through June 13, 2012. During his stay in Dr. Karim’s lab, he received hands-on training in the use of standard molecular biology tools for detection of tick-borne pathogens, as well as other techniques useful in vector molecular biology. He also gained valuable experience working with Dr. Karim and his students on analyzing samples collected as part of this project. While Dr. Iqbal was in the United States, his colleagues back at UAF launched a new graduate course on Physiology of Parasites, which was presented during the March-July semester. The participants in the course included four UAF graduate students working on this project. Thanks to this training and to the new equipment installed in the Molecular Parasitology Lab at UAF with funds from this project, the students are currently focusing on collecting and processing tissue samples. Linkages have been established with the University of Balochistan in Quetta, the University of Sindh in Jamshoro, and a network of veterinarians in Kashmir to expand the scope of the sample collection activities.
The electrical power supply at UAF remains unreliable, so a backup generator was recently purchased with supplemental funds from the university in order to ensure that tissue samples stored in the lab’s ultra-low temperature freezer are properly maintained. Besides ongoing sample collection and analytical efforts, the UAF team’s plans for the coming months include an international conference on parasitic threats to livestock (tentatively planned for November-December 2012) and a hands-on training workshop on molecular detection of parasitic diseases (tentatively planned for January 2013). Dr. Karim also expects to host another Pakistani visiting scientist for training at USM in January-March 2013.
2013 Show summary || Hide summary
During spring, the project’s activities focused on quantification of Rickettsia amblyommii in ticks, investigation of bacterial diversity in ticks infesting Pakistani livestock, and further study on the infection of Theileria annulata in Hyalomma species. One female graduate assistant and four male undergraduate research interns have been recruited to support project staff to perform field work. On the Pakistani side, a new graduate level course titled “Physiology of Parasites” was offered by Dr. Iqbal’s team at UAF during the spring semester and 5 graduate students have enrolled in this course. Two graduate students have also joined Dr. Iqbal’s team to work on their doctoral thesis. Ms. Asma Kausar, a visiting student from UAF, has arrived at USM on March 16 to start taking the training on molecular techniques.
Although the release of the Year 2 funding delayed the purchase of some key equipment, the Pakistani team finally received one of the equipment, Nanodrop Spectrophotometer, in January. Other equipments have also been ordered. A new clean room has been constructed for pathogen detection reactions at UAF. Four research papers have been published and six presentations were made at conferences and workshops during the 1st quarter of 2013.
Another graduate student Ms. M. Jawad-ul-Hassan is currently waiting for her visa, and is expected to arrive at UMS for training in May 2013. For the upcoming months, the project team will conduct characterization of tick salivary genes using RNA interference as well as the molecular detection of tick-borne infectious agents in ticks infesting Pakistani livestock. The team is also planning a hands-on training workshop in “molecular detection of tick and tick-borne pathogens”, to be held at the end of 2013 after the return of UAF scholars from Dr. Karim’s lab.
In the summer, collection of tick samples expanded from Kashmir, Sindh, and Punjab to the outlying provinces of Baluchistan and KPK. Samples were shipped to Dr. Karim’s lab at USM for species identification and analysis. 112 animal blood samples were screened for the presence of a tick-borne cattle parasite (Theileria annulata) known to adversely affect 250 million animals world-wide and 177 ticks (Hyalomma) were tested for the presence of the parasite. These screening results have shown great risk of theileriosis and babesiosis in Pakistan. Using tick saliva and proteins from the parasite (T. annulata), the researchers plan to develop a vaccine.
The US team has also constructed normalized cDNA libraries from Amblyomma maculatum synganglia (brain) and Amblyomma tuberculatum salivary glands. The data set is being annotated so that the team can get a snapshot of gene expression for further computational analysis. Meantime, illumine sequencing (RNA-seq) approach was used to get a snapshot of tick sialotranscriptome, and these data will be annotated for publication.
At UAF, the research team has been offering a graduate upper level course “Physiology of Parasites” which was attended by five students this semester. Four undergraduate students continue working on this project in the lab as well as in the field. Four students (one male Ph.D. student in UAF, one male graduate student, and two female graduate students in USM) defended their thesis related to this research project. Ms. Asma Kausar, a visiting student from UAF, has been taking the training on molecular techniques and plans to return to Pakistan on September 15. Two graduate students (Mr. Rao Muhammad Siddique and Ms. Qurat ul Ain) have joined the project to study the functional genomics of the ticks for their graduate research.
Upon the receiving of Year 2 funding, a few more equipment have been purchased. Those equipment include the Biological Safety cabinet (Type II), Sonicator, Ice Maker, Mini Centrifuge, water deionizer, and bench top autoclave.
Due to the energy crisis in Pakistan, the lab equipment has to be powered by power generator, which made it difficult to work on the bench. The travel budget on the Pakistani side became very tight because of the inflation as well as the increased international airfare, so the US team had to stretch to train the two visiting scholars. The two Pakistani graduate students who will be trained in the US lab had experienced delays in receiving their visa (one student just received his visa on July 2, and the other is still waiting.)
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