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Author: Donna Huber

Limited genetic variability of Cytauxzoon felis apical membrane antigen-1 (ama1) from domestic cats and bobcats

BACKGROUND:

Cytauxzoon felis is a tick-transmitted apicomplexan that causes cytauxzoonosis in domestic cats (Felis catus). Even with intensive care, the mortality rate of acute cytauxzoonosis approaches 40% in domestic cats, while bobcats (Lynx rufus), the natural intermediate host of C. felis, remain clinically asymptomatic. However, multiple reports of domestic cats surviving acute disease without any treatment exist. One hypothesis for survival of these cats is infection with unique C. felis genotypes of lower pathogenicity. Prior studies have identified genetically distinct C. felis isolates containing polymorphisms within internal transcribed spacer regions (ITS) of the rRNA operon. However, these polymorphisms do not correlate with the clinical outcome of cytauxzoonosis, and so additional genetic markers are needed to test this hypothesis. We selected C. felis apical membrane antigen-1 (ama1) as a potential genetic marker of differential pathogenicity. AMA1 is a vaccine candidate for relatives of C. felis within Plasmodium spp.; however its historically high level of genetic polymorphism has resulted in escape from vaccine-induced immunity. While such diversity has hindered vaccine development, the expected polymorphism within the ama1 gene may be useful to evaluate population genetics.

RESULTS:

A 677 bp sequence of the C. felis ama1 gene was PCR-amplified from 84 domestic cats and 9 bobcats and demonstrated 99.9% sequence identity across all samples. A single nucleotide polymorphism (SNP) was identified in domestic cats and bobcats with evidence for co-infection with both genotypes identified in two domestic cats. The prevalence of the two genotypes varied with geographical distribution in domestic cats. Nucleotide diversity (π) and haplotype diversity (H) were calculated for C. felis ama1 and ama1 of related apicomplexans to assess genetic diversity. Based on these values (π = 0.00067 and H = 0.457), the diversity of the C. felis ama1 gene region analyzed is considerably lower than what is documented in related apicomplexans.

CONCLUSIONS:

In surprising contrast to related apicomplexans, our results support that the sequence of the C. felis ama1 gene is highly conserved. While lack of genetic diversity limits utility of C. felis AMA1 as a genetic marker for clinical outcome, it supports further investigation as a vaccine candidate for cytauxzoonosis.

Jaime L. Tarigo, Lisa S. Kelly, Holly M. Brown and David S. Peterson. 2019. Parasites & Vectors; 12:115. https://doi.org/10.1186/s13071-019-3347-5

Genome Editing by CRISPR/Cas9 in Trypanosoma cruzi

The genetic manipulation of the human parasite Trypanosoma cruzi has been significantly improved since the implementation of the CRISPR/Cas9 system for genome editing in this organism. The system was initially used for gene knockout in T. cruzi, later on for endogenous gene tagging and more recently for gene complementation. Mutant cell lines obtained by CRISPR/Cas9 have been used for the functional characterization of proteins in different stages of this parasite’s life cycle, including infective trypomastigotes and intracellular amastigotes. In this chapter we describe the methodology to achieve genome editing by CRISPR/Cas9 in T. cruzi. Our method involves the utilization of a template cassette (donor DNA) to promote double-strand break repair by homologous directed repair (HDR). In this way, we have generated homogeneous populations of genetically modified parasites in 4–5 weeks without the need of cell sorting, selection of clonal populations, or insertion of more than one resistance marker to modify both alleles of the gene. The methodology has been organized according to three main genetic purposes: gene knockout, gene complementation of knockout cell lines generated by CRISPR/Cas9, and C-terminal tagging of endogenous genes in T. cruzi. In addition, we refer to the specific results that have been published using each one of these strategies.

 

Noelia Lander, Miguel A. Chiurillo, Roberto Docampo. 2019. Methods Mol Biol. 2019;1955:61-76. doi: 10.1007/978-1-4939-9148-8_5

Serological proteomic screening and evaluation of a recombinant egg antigen for the diagnosis of low-intensity Schistosoma mansoni infections in endemic area in Brazil

BACKGROUND:

Despite decades of use of control programs, schistosomiasis remains a global public health problem. To further reduce prevalence and intensity of infection, or to achieve the goal of elimination in low-endemic areas, there needs to be better diagnostic tools to detect low-intensity infections in low-endemic areas in Brazil. The rationale for development of new diagnostic tools is that the current standard test Kato-Katz (KK) is not sensitive enough to detect low-intensity infections in low-endemic areas. In order to develop new diagnostic tools, we employed a proteomics approach to identify biomarkers associated with schistosome-specific immune responses in hopes of developing sensitive and specific new methods for immunodiagnosis.

 

METHODS AND FINDINGS:

Immunoproteomic analyses were performed on egg extracts of Schistosoma mansoni using pooled sera from infected or non-infected individuals from a low-endemic area of Brazil. Cross reactivity with other soil-transmitted helminths (STH) was determined using pooled sera from individuals uniquely infected with different helminths. Using this approach, we identified 23 targets recognized by schistosome acute and chronic sera samples. To identify immunoreactive targets that were likely glycan epitopes, we compared these targets to the immunoreactivity of spots treated with sodium metaperiodate oxidation of egg extract. This treatment yielded 12/23 spots maintaining immunoreactivity, suggesting that they were protein epitopes. From these 12 spots, 11 spots cross-reacted with sera from individuals infected with other STH and 10 spots cross-reacted with the negative control group. Spot number 5 was exclusively immunoreactive with sera from S. mansoni-infected groups in native and deglycosylated conditions and corresponds to Major Egg Antigen (MEA). We expressed MEA as a recombinant protein and showed a similar recognition pattern to that of the native protein via western blot. IgG-ELISA gave a sensitivity of 87.10% and specificity of 89.09% represented by area under the ROC curve of 0.95. IgG-ELISA performed better than the conventional KK (2 slides), identifying 56/64 cases harboring 1-10 eggs per gram of feces that were undiagnosed by KK parasitological technique.

 

CONCLUSIONS:

The serological proteome approach was able to identify a new diagnostic candidate. The recombinant egg antigen provided good performance in IgG-ELISA to detect individuals with extreme low-intensity infections (1 egg per gram of feces). Therefore, the IgG-ELISA using this newly identified recombinant MEA can be a useful tool combined with other techniques in low-endemic areas to determine the true prevalence of schistosome infection that is underestimated by the KK method. Further, to overcome the complexity of ELISA in the field, a second generation of antibody-based rapid diagnostic tests (RDT) can be developed.

 

Vanessa Silva-Moraes, Lisa Marie Shollenberger, William Castro-Borges, Ana Lucia Teles Rabello, Donald A. Harn, Lia Carolina Soares Medeiros, Wander de Jesus Jeremias, Liliane Maria Vidal Siqueira, Caroline Stephane Salviano Pereira, Maria Luysa Camargos Pedrosa, Nathalie Bonatti Franco Almeida, Aureo Almeida, Jose Roberto Lambertucci, Nídia Francisca de Figueiredo Carneiro, Paulo Marcos Zech Coelho, Rafaella Fortini Queiroz Grenfell. 2019. PLOS Neglected Tropical Diseases.
https://doi.org/10.1371/journal.pntd.0006974

Register Now! Molecular Parasitology & Vector Biology Symposium

Symposium 2019 announcement

Registration is now open for the 29th Annual Molecular Parasitology & Vector Biology Symposium hosted by the Center for Tropical and Emerging Global Diseases. It will be held on Wednesday, May 1, 2019, at The Georgia Center on the University of Georgia campus in Athens, GA.

This day-long regional conference on parasites and host/parasite interaction draws more than 200 attendees from many departments at UGA and colleagues from other institutions throughout the United States.

As CTEGD is celebrating its 20th anniversary, 4 outstanding alumni have been invited to give spotlight presentations throughout the day in addition to the oral presentations from graduate students, postdocs, and senior researchers.

 

Invited Alumni Speakers:

 

  • James Morris, mentored by Kojo Mensa-Wilmot, is a professor in the Department of Genetics and Biochemistry at Clemson University.

 

  • Matthew Collins, mentored by Rick Tarleton, is an assistant professor in the Division of Infectious Diseases at Emory University School of Medicine

 

  • Tiffany Weinkopff, mentored by Patrick Lammie, is an assistant professor in the Department of Microbiology and Immunology at the University of Arkansas’s College of Medicine.

 

  • Marc-Jan Gubbels, mentored by Boris Striepen, is a professor in the Department of Biology at Boston College.

 

Oral and Poster Presentations

Graduate students, postdoctoral fellows, and other researchers are invited to present their research in either an oral or poster presentation. Abstracts for these presentations are due by April 12. During registration, please select Poster or Speak. An email will be sent to you with instructions on submitting your abstract. Please keep the following in mind when preparing your submission:

 

  • All abstracts must be submitted online by April 12, 2019, via the link in the email you received after registering.
  • Abstracts should include Title, Author(s), and Affiliation(s).
  • Abstracts should be 300 words or less, excluding title, author(s), and affiliation(s).
  • Notification of submission will be emailed to you.
  • Note: there has been issues of special characters (i.e. α and γ) not displaying in the submitted abstract. Therefore, please note the use of special characters by spelling out the word next to the symbol, e.g. IFN-γ (gamma) or β (Beta). The correction will be made during the editing process.
  • Abstracts will be published online by Wednesday, April 24.

 

Cost & Register

There is no cost to attend the Symposium or the full catered lunch, but registration is required.

Hotel Rooms: For those wishing to stay overnight, reservations can be made at The Georgia Center Hotel. The Holiday Inn is also nearby.

 

Register here: http://register.ctegd.uga.edu/

For more information: https://ctegd.uga.edu/events/symposium/

Further insights of selenium-containing analogues of WC-9 against Trypanosoma cruzi

Graphical abstract

As a continuation of our project aimed at searching for new chemotherapeutic agents against American trypanosomiasis (Chagas disease), new selenocyanate derivatives were designed, synthesized and biologically evaluated against the clinically more relevant dividing form of Trypanosoma cruzi, the etiologic agent of this illness. In addition, in order to establish the role of each part of the selenocyanate moiety, different derivatives, in which the selenium atom or the cyano group were absent, were conceived, synthesized and biologically evaluated. In addition, in order to study the optimal position of the terminal phenoxy group, new regioisomers of WC-9 were synthesized and evaluated against T. cruzi. Finally, the resolution of a racemic mixture of a very potent conformationally rigid analogue of WC-9 was accomplished and further tested as growth inhibitors of T. cruzi proliferation. The results provide further insight into the role of the selenocyanate group in its antiparasitic activity.

 

María N. Chao, María V. Lorenzo-Ocampo, Sergio H. Szajnman, Roberto Docampo, Juan B. Rodriguez. 2019. Bioorganic & Medicinal Chemistry. https://doi.org/10.1016/j.bmc.2019.02.039

Prime-Boost Vaccine Regimen for SjTPI and SjC23 Schistosome Vaccines, Increases Efficacy in Water Buffalo in a Field Trial in China

Schistosomiasis remains a serious zoonotic disease in China and the Philippines. Water buffalo and cattle account for the majority of transmission. Vaccination of water buffalo is considered a key strategy to reduce disease prevalence. Previously, we showed that vaccination of water buffalo with SjC23 or SjCTPI plasmid DNA vaccines, induced 50% efficacy to challenge infection. Here, we evaluated several parameters to determine if we can develop a two dose vaccine that maintains the efficacy of the three dose vaccine. We performed four trials evaluating: (1) lab produced vs. GLP grade vaccines, (2) varying the time between prime and boost, (3) the influence of an IL-12 adjuvant, and (4) a two dose heterologous (DNA-protein) prime-boost. We found the source of the DNA vaccines did not matter, nor did increasing the interval between prime and boost. Elimination of the IL-12 plasmid lowered homologous DNA-DNA vaccine efficacy. A major finding was that the heterologous prime boost improved vaccine efficacy, with the prime-boost regimen incorporating both antigens providing a 55% reduction in adult worms and 53% reduction in liver eggs. Vaccinated buffalo produced vaccine-specific antibody responses. These trials suggest that highly effective vaccination against schistosomes can be achieved using a two dose regimen. No adjuvants were used with the protein boost, and the potential that addition of adjuvant to the protein boost to further increase efficacy should be evaluated. These results suggest that use of these two schistosome vaccines can be part of an integrated control strategy to reduce transmission of schistosomiasis in Asia.

 

Akram A. Da’Dara, Changlin Li, Xinling Yu, Mao Zheng, Jie Zhou, Lisa M. Shollenberger, Yue-sheng Li and Donald A. Harn. 2019. Front. Immunol. https://doi.org/10.3389/fimmu.2019.00284

Targeted Inhibition of Plasmodium falciparum Calcium-Dependent Protein Kinase 1 with a Constrained J Domain-Derived Disruptor Peptide

graphical abstract

To explore the possibility of constrained peptides to target Plasmodium-infected cells, we designed a J domain mimetic derived from Plasmodium falciparum calcium-dependent protein kinase 1 ( PfCDPK1) as a strategy to disrupt J domain binding and inhibit PfCDPK1 activity. The J domain disruptor (JDD) peptide was conformationally constrained using a hydrocarbon staple and was found to selectively permeate segmented schizonts and colocalize with intracellular merozoites in late-stage parasites. In vitro analyses demonstrated that JDD could effectively inhibit the catalytic activity of recombinant PfCDPK1 in the low micromolar range. Treatment of late-stage parasites with JDD resulted in a significant decrease in parasite viability mediated by a blockage of merozoite invasion, consistent with a primary effect of PfCDPK1 inhibition. To the best of our knowledge, this marks the first use of stapled peptides designed to specifically target a Plasmodium falciparum protein and demonstrates that stapled peptides may serve as useful tools for exploring potential antimalarial agents.

Briana R. Flaherty, Tienhuei G. Ho, Sven H. Schmidt, Friedrich W. Herberg, David S. Peterson, and Eileen J. Kennedy. 2019. ACS Infectious Diseases. DOI: 10.1021/acsinfecdis.8b0034

$1.5 million initiative to upgrade labs across campus

Michael Strand
A $1.5 million initiative to upgrade labs across campus is enabling faculty members such as Regents Professor Michael Strand to enhance their research productivity. (Photo by Dorothy Kozlowski/ UGA)

Athens, Ga. – Labs and research support spaces across campus will be getting an upgrade, thanks to a $1.5 million presidential initiative that seeks to build on the university’s dramatic growth in research activity.

Presidential renovation funds have been distributed to nine schools and colleges and will be used to upgrade labs and replace core equipment that enables faculty members to conduct research and be more competitive in seeking grant funding. Proposals were solicited from deans and chosen based on links to college and university strategic priorities, as well as implications for faculty recruitment efforts and grant funding opportunities.

 

“To advance the research mission of the university and attract and retain outstanding faculty, we must support state-of-the-art facilities that assist the faculty with their groundbreaking work,” said President Jere W. Morehead. “I am pleased the institution has been able to help several faculty with critical needs, thanks to this initiative.”

In the College of Agricultural and Environmental Sciences, an upgrade to an insectary that will be used to rear mosquitoes will enable Regents’ Professor and National Academy of Sciences member Michael Strand and several of his colleagues in the department of entomology to expand their research on infectious diseases such as malaria and dengue fever. “We’re going to be able to do a whole series of experiments that we currently can’t do,” Strand said, adding that the upgraded facility opens up new opportunities for grants.

Upgrades to the Sensory Evaluation and Product Development Lab in the College of Family and Consumer Sciences will enable assistant professor Ginnefer Cox to develop and evaluate new food product formulations more efficiently while also giving students hands-on experiences and facilitating industry partnerships. “This new space is going to have equipment that helps train students to be the next product developers,” Cox said. “The upgrades also create more opportunities to collaborate in research with food companies, which opens up opportunities for students to interact with them and obtain internships and permanent employment.”

In the department of physics and astronomy, part of the Franklin College of Arts and Sciences, renovation funds will aid in faculty recruitment by modernizing an outdated laboratory. “We’re really excited to have received this funding,” said department head Phillip Stancil. “The space has been unused for the last several years, and with this renovation it’ll be ready for a new experimentalist to move in.”

Other schools and colleges that have received funding through presidential renovation funds are the College of Engineering, College of Environment and Design, Odum School of Ecology, College of Public Health, College of Veterinary Medicine and the Warnell School of Forestry and Natural Resources.

Interim Senior Vice President for Academic Affairs and Provost Libby V. Morris noted that the lab renovation funds come at a time when sponsored research awards have increased by 34 percent over the past five years. It also coincides with recruitment initiatives that will bring up to 25 new faculty members to campus.

 

“Research activity at the University of Georgia has grown significantly in recent years, with strategic investments in faculty and facilities enabling discoveries that point the way to a healthier and more promising future,” Morris said.

Writer: Sam Fahmy, 706-583-0727, sfahmy@uga.edu

Priority use cases for antibody-detecting assays of recent malaria exposure as tools to achieve and sustain malaria elimination

Measurement of malaria specific antibody responses represents a practical and informative method for malaria control programs to assess recent exposure to infection. Technical advances in recombinant antigen production, serological screening platforms, and analytical methods have enabled the identification of several target antigens for laboratory based and point-of-contact tests. Questions remain as to how these serological assays can best be integrated into malaria surveillance activities to inform programmatic decision-making. This report synthesizes discussions from a convening at Institut Pasteur in Paris in June 2017 aimed at defining practical and informative use cases for serology applications and highlights five programmatic uses for serological assays including: documenting the absence of transmission; stratification of transmission; measuring the effect of interventions; informing a decentralized immediate response;  and testing and treating P. vivax hypnozoite carriers.

Greenhouse B, Daily J, Guinovart C, Goncalves B, Beeson J, Bell D, Chang MA, Cohen JM, Ding X, Domingo G, Eisele TP, Lammie PJ, Mayor A, Merienne N, Monteiro W, Painter J, Rodriguez I, White M, Drakeley C, Mueller I, Malaria Serology Convening. 2019. Gates Open Res.; doi: 10.12688/gatesopenres.12897.1. eCollection 2019.

Celebrate the 10th Anniversary of SEFCIG

SECFIG annual meeting

Celebrate the 10th anniversary of the Southeast Flow Cytometry Interest Group (SEFCIG) with 4 days of educational training and scientific talks. SEFCIG was founded by Julie Nelson, the director of UGA’s CTEGD Cytometry Shared Resource Laboratory. The 10th Annual Meeting is being hosted on the University of Georgia campus in Athens, GA.

10th Annual Meeting March 5 – 8, 2019

March 5 – 6         ExCyte – Flow Cytometry Boot Camp

Register here: https://excyteatuga.eventbrite.com/

Special discounts are available to the UGA community and those attending SEFCIG, please email Tim Bushnell (timbushnell@expertcytometry.com) for details.

March 7 – TechnoFlow

Location: Paul D. Coverdell Building Room S175

11:00 – 12:00

Forensic Flow – Join us to test your skills at detecting bad flow data
Jodi Kroeger, Moffitt Cancer Institute

12:00 – 1:00

 Luncheon – Coverdell Rotunda

1:00 – 1:45

Multi-Dimensional Functional Profiling of Human Rhinovirus and Allergen- specific T-Cells By Means Of Spectral Flow Cytometry
Liesbeth Paul and Joanne Lannigan, University of Virginia

1:45 – 2:30

 Imaging and Spectral Cytometry go Viral!
Joanne Lannigan, University of Virginia

2:30 – 3:15

High Dimensional Mass Cytometry Data Analysis
Deon Bryant, Emory University

3:15 – 4:00

Next Generation Cell Sorting: New Technologies and Strategies
Joe Trotter, Becton Dickinson

6:00 – 9:00

Opening Reception – Pecan Tree Galleria

March 8 – General Session

Location: Masters Hall, Georgia Center for Continuing Education

Flow in the South

9:00 – 9:10

Welcome and Introductions

9:10 – 9:55

Canine Breast Cancer Immunotherapy as a Model of Human Disease
Curtis Bird, Auburn University

9:55 – 10:40

Single cell analyses of human B cell responses: Lessons from infectious disease and autoimmunity
Jens Wrammert, Emory University

10:40 – 11:25

Using Flow Cytometry to Catch Parasites Sleeping
Dennis Kyle, University of Georgia

11:25 – 12:00

Exhibits

Vendor Break Out Session with Box Lunches

12:00 – 2:45

Vendor Talks and Exhibits

Flow in the World

2:45 – 3:30

Extracellular Vesicle-Biome Analysis by Nanoscale High Resolution Flow Cytometry
Terry Morgan, Oregon School of Health

3:30 – 4:15

Optimizing and validating FC-based EV measurements
John Nolan, Scintillon Institute

4:15 – 5:00

Developing Flow Cytometry Assays to Support Clinical Trials
Jake Jacobberger, Case Western

Register and make hotel reservations at www.sefcig.org